RU2840049C2 - Novel molecules of agonistic anti-tnfr2 antibodies - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to molecular biology and medicine, in particular to novel antibody molecules that specifically bind to TNFR2 on a target cell and are thereby agonists to TNFR2, but do not block the TNF-α ligand from binding with TNFR2. Also described is the use of such antibody molecules in medicine, for example, in the treatment of cancer or chronic inflammatory diseases.

EFFECT: disclosed antibodies are powerful therapeutic agents and are useful in medicine.

37 cl, 22 dwg, 10 tbl, 7 ex

Description

Field of technology to which the invention relates

The present invention relates to new agonistic antibodies that specifically bind to tumor necrosis factor receptor 2 (TNFR2) but do not block the TNF-α ligand from binding to TNFR2. The invention also relates to their use in medicine, for example for the treatment of cancer or chronic inflammatory diseases.

Prerequisites for the creation of an invention

Tumor necrosis factor (TNF) receptor 2 (TNFR2, TNFR-2, or TNFRII), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) and CD120b, is a membrane receptor that binds tumor necrosis factor alpha (TNF-α or TNFα). It is found, for example, on the surface of T cells, monocytes, and macrophages, and can activate the proliferation of TNFR2-expressing cells via nuclear factor kappa B (NF-κB). Notably, TNFR2 is highly up-regulated in cancer and, in particular, on tumor-infiltrating immune cells, such as regulatory T cells (Tregs), CD8 ⁺ cytotoxic effector T cells, and various myeloid cell subsets

TNFR2 has been discussed as a promising target for cancer immunotherapy and has been described to be highly expressed on the surface of, among others, intratumoral Tregs and many human tumor cells (Williams GS et al, Oncotarget. 2016; 7(42): 68278–68291; Vanamee ES et al, Trends in Molecular Medicine, 2017, vol. 23, issue 11, 1037–1046; Frontiers in Immunology , November 2017 | Volume 8 | Article 1482, Sci Signal. 2018 Jan 2;11(511)).

Regulatory T cells (which may also be called Treg cells, Tregs, or _Tregs , and which were formerly known as suppressor T cells or suppressive regulatory T cells) are a subset of T cells that are capable of suppressing other immune cells under normal and pathological immune conditions. Tregs are CD4-positive cells (CD4 ⁺ cells). There are other CD4 ⁺ T cells that are not Tregs; however, Tregs can be distinguished from non-Treg CD4 ⁺ cells in that Tregs are also FOXP3-positive (FOXP3 ⁺ ), whereas non-Treg CD4 ⁺ cells are FOXP3-negative ( ^FOXP3- ). Tregs can also be distinguished from non-Treg CD4 ⁺ cells, in that Tregs are also CD25 ⁺ CD127 ^neg/low , while non-Treg CD4 ⁺ cells are either CD25 ^- CD127 ⁺ or CD25 ⁺ CD127 ⁺ .

TNFR2 has also been discussed in relation to autoimmune diseases (Faustman DL et al, Front Immunol. 2013; 4: 478, Clin Transl Immunology. 2016 Jan 8; 5(1): J Neurosci. 2016 May 4; 36(18):5128-43) and inflammatory diseases (Ait-Ali D et al, Endocrinology. 2008 Jun; 149(6):2840-52, Sci Rep. 2016 Sep 7; 6:32834).

Anti-TNFR2 antibodies of different types and with different characteristics have also been described previously. For example, Williams et al (Oncotarget. 2016 Oct 18; 7(42):68278-68291) describe both ligand-blocking and non-ligand-blocking agonist antibodies.

WO 2014/124134 describes the use of a TNFR2 agonist, such as an agonistic anti-TNFR2 antibody and/or an NF-κB activator, for the in vitro production of a composition enriched in CD4+CD25 ^hi Tregs. The composition is stated to be useful for the treatment of immunological disorders or infectious diseases in patients.

WO 2017/040312 describes anti-TNFR2 antibodies and, in particular, agonist anti-TNFR2 antibodies that are capable of stimulating TNFR2 signaling and influencing the expansion or proliferation of Tregs. WO 2017/040312 describes antibodies that specifically bind to an epitope comprising the KCSPG sequence, but not to an epitope comprising the KCRPG sequence, thereby excluding the antibodies of US Patent 9,821,010 described above, or, alternatively, do not bind to another member of the TNFR superfamily. The agonist antibodies are claimed to be useful in the treatment of immunological diseases. WO 2017/040312 further describes the complete sequence of human TNFR2.

WO 2017/083525 discusses pharmacological compositions comprising anti-TNFR2 antibodies and their use in the treatment of TNF-α and/or TNFR2-associated disorders, such as cancer. WO 2017/083525 further discusses antibodies comprising a human IgG1 Fc domain that is null for binding to the Fcγ receptor, as well as for suppressing Treg expansion.

In addition, anti-TNFR2 antibodies that are capable of acting as TNFR2 agonists have been described by Galloway et al. (Eur. J. Immunol. 22:3045–3048, 1992), Tartaglia et al. (J. Biol. Chem. 268:18542–18548, 1993), Tartaglia et al. (J. Immunol. 151:4637–4641, 1993), Smith et al. (J. Biol. Chem. 269:9898–9905, 1994), and Amrani et al. (Am. J. Respir. Cell. Mol. Biol. 15:55–63, 1996).

However, none of these papers describe or propose agonist TNFR2 antibodies that specifically bind to TNFR2 but do not block TNF-α ligand from binding to the same TNFR2.

Fc receptors are membrane proteins found on the cell surface of immune effector cells, including monocytes, macrophages, dendritic cells, neutrophils, mast cells, basophils, eosinophils, as well as natural killer cells and B lymphocytes. The name derives from their binding specificity to the Fc region of antibodies. Fc receptors are found on the cell membrane, also known as the plasma membrane or cytoplasmic membrane. Fc receptors can be divided into activating FcγR and inhibitory FcγR, which are known to coordinate cellular activation by binding aggregated immunoglobulin G Fc and transmitting activating or inhibitory signals to the cell via intracellular ITAM or ITIM motifs. FcR binding of aggregated immunoglobulin or immune complexes can mediate internalization of the antibody into the cell and can result in antibody-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity, or antigen presentation or antigen cross-presentation. FcRs are also known to mediate or enhance cross-linking of cell surface receptors bound by antibodies. Such cross-linking is known to be required for some (Li et al 2011. 'Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies', Science, 333: 1030-4.; White et al. 2011. 'Interaction with FcgammaRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody', J Immunol, 187: 1754-63 ) but not all (Richman et al 2014. 'Anti-human CD40 monoclonal antibody therapy is potent without FcR crosslinking', Oncoimmunology, 3: e28610) of the antibodies' ability to activate signaling in target cells and may or may not be required to achieve therapeutic effects.

A subgroup of Fc receptors are the Fcγ receptors (Fc gamma receptors, FcgammaR, FcγR), which are specific for IgG antibodies. There are two types of Fcγ receptors: activating Fcγ receptors (also referred to as Fcγ activation receptors) and inhibitory Fcγ receptors. Activating and inhibitory receptors transmit their signals through immunoreceptor tyrosine-based activation motifs (ITAMs) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs), respectively. In humans, FcγRIIb (CD32b) is the inhibitory Fcγ receptor, whereas FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), and FcγRIIIa (CD16a) activate Fcγ receptors. FcγgRIIIb is a GPI-linked receptor expressed on neutrophils that lacks an ITAM motif, but due to its ability to cross-link lipid rafts and interact with other receptors, it is also considered an activator. In mice, the activating receptors are FcγRI, FcγRIII, and FcγRIV.

It is well known that antibodies can modulate immune cell activity through interactions with Fcγ receptors. In particular, how antibody immune complexes modulate immune cell activation is determined by their relative binding to activating and inhibitory Fcγ receptors. Different antibody isotypes bind to activating and inhibitory Fcγ receptors with different affinities, resulting in different A:I ratios (activation to inhibition ratios) (Nimmerjahn et al; Science. 2005 Dec 2;310(5753):1510-2).

By binding to inhibitory Fcγ receptors, the antibody can inhibit, block and/or reduce the functions of effector cells. By binding to inhibitory FcγR, antibodies can further stimulate cell activation through aggregation of antibody-targeted signaling receptors on the target cell (Li et al. 2011. 'Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies', Science, 333: 1030-4; White et al 2011. 'Interaction with FcgammaRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody', J Immunol, 187: 1754-63; White et al 2014. 'Fcgamma receptor dependency of agonistic CD40 antibody in lymphoma therapy can be overcome through antibody multimerization', J Immunol, 193: 1828-35).

By binding to the activating Fcγ receptor, an antibody can activate effector cell functions and thereby trigger mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine release and/or antibody-dependent endocytosis, and NETosis (i.e., activation and release of neutrophils, neutrophil extracellular traps) in the case of neutrophils. Binding of antibodies to the activating Fcγ receptor can also lead to an increase in certain activation markers such as CD40, MHCII, CD38, CD80, and/or CD86.

Recent data published by the present inventors, among others, demonstrate a critical and differential dependence of CD8 T cell agonist and Treg-depleting anti-4-1BB antibodies on binding to activating and inhibitory FcγRs, respectively, on therapeutic efficacy (Buchan et al., 'Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function', Immunity 2018 49(5):958–970). Moreover, importantly, co-administration of CD8 T cell agonist and Treg-depleting anti-4-1BB antibodies optimized for binding to activating and inhibitory FcγRs, respectively, impaired therapeutic activity. These data demonstrate the critical importance of designing antibodies with appropriate and tailored engagement of activating and inhibitory FcγRs to maximize the therapeutic activity of antibodies with different mechanisms of action. At the same time, they demonstrate that suboptimal engagement of activating and inhibitory FcγRs may seriously compromise therapeutic efficacy.

These data were unexpected because they contrasted with data on antibodies to other TNFSR members, particularly immunostimulatory anti-CD40 antibodies, which show an obligatory requirement for the engagement of an inhibitory but not activating FcγR (Li et al. 2011. 'Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies', Science, 333: 1030-4; White et al. 2011. 'Interaction with FcgammaRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody', J Immunol, 187: 1754-63). Taken together, these results demonstrate that FcγR dependence can vary between antibodies to different targets of the same receptor superfamily, and even between different types of antibodies to the same target, in a way that is difficult to predict but may be critical to understand and exploit in the development of antibodies for therapeutic use.

The essence of the invention

In the course of work leading to this invention, as well as a parallel invention, two major distinct groups of anti-TNFR2 antibodies with potent therapeutic effects and distinct characteristics and mechanisms of action were identified.

The inventors have identified for the first time the potent therapeutic activity of antagonist anti-TNFR2 antibodies that block TNF-α binding to the TNFR2 receptor. It has been demonstrated that the activity of such antibodies is dependent on FcγR interactions and, in particular, on binding to an activating FcγR for therapeutic activity in vivo. It has been found that this group or category of potent therapeutic anti-TNFR2 reagents are characterized by 1) potent blocking and inhibition of TNF-α-induced TNFR2 signaling, and 2) FcγR interaction-dependent activity, with the strongest benefit from interaction with activating versus inhibitory FcγRs.

The inventors then identified a distinct group of anti-TNFR2 antibodies with similarly potent in vivo therapeutic activity, but whose characteristics are in many respects the opposite of those of the antagonist, blocking TNFR2 antibody types that comprise the first group. The anti-TNFR antibodies of this second group do not rely on TNF-α blockade or inhibition of TNFR2 signaling for therapeutic activity, but rather are characterized by potent activation of TNFR2 signaling. Furthermore, unlike the blocking antibodies of the first group, the agonist antibodies of the second group do not exhibit an obligate dependence on antibody:FcγR interaction, even though their activity is enhanced with FcγR:interacting antibody variants. Furthermore, unlike the antagonist blocking antibodies of the first group, the agonist antibodies of the second group exhibit greatest activity in antibody variants with improved binding to inhibitory versus activating FcγR.

The present invention relates to a second group of anti-TNFR2 antibodies, i.e., agonist antibody molecules that specifically bind to TNFR2 but do not block the TNF-α ligand from binding to TNFR2. Such antibodies are potent therapeutic reagents and are useful in medicine.

Antagonist blocking antibodies belonging to the first group are used in the following examples for comparison with the agonist non-blocking TNFR2 antibody molecules of the present invention. In the examples for comparison, other antibodies with some characteristics similar to those of the first or second group, or both, are also used, as further explained below.

Thus, the present invention relates to agonist antibody molecules that specifically bind to TNFR2 on a target cell and do not block TNF-α ligand binding to TNFR2.

The present invention also relates to specific examples of such novel TNFR2 agonist antibody molecules.

The present invention also relates to isolated nucleotide sequences encoding at least one of the above antibody molecules.

The present invention also relates to plasmids comprising at least one of the above nucleotide sequences.

The present invention also relates to viruses containing at least one of the above nucleotide sequences or plasmids.

The present invention also relates to cells containing at least one of the above nucleotide sequences, or at least one of the above plasmids, or at least one of the above viruses.

The present invention also relates to the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses and/or cells for use in medicine.

The present invention also relates to the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses and/or cells for use in the treatment of cancer or a chronic inflammatory disease.

The present invention also relates to the use of the above antibody molecules, nucleotide sequences, plasmids, viruses and/or cells for use in the treatment of cancer or a chronic inflammatory disease.

The present invention also relates to pharmaceutical compositions comprising or consisting of at least one of the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses and/or cells and, optionally, a pharmaceutically acceptable diluent, carrier or excipient. Such a pharmaceutical composition can be used in the treatment of cancer or a chronic inflammatory disease.

In addition, the present invention relates to methods of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one of the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses and/or cells.

The present invention also relates to antibody molecules, antibody molecules for use, isolated nucleotide sequences, isolated nucleotide sequences for use, plasmids, plasmids for use, viruses, viruses for use, cells, cells for use, uses, pharmaceutical compositions and methods of treatment as described herein with reference to the accompanying description, examples and/or figures.

Detailed description of the invention

Thus, the present invention relates to TNFR2 agonist antibody molecules that specifically bind to TNFR2 but do not block the TNF-α ligand from binding to the same TNFR2. Preferably, the antibody molecules have intrinsic agonist activity.

The agonist antibody molecules described herein do not block TNF-α from binding to TNFR2, nor do they block TNFR2 signaling. It has been clearly demonstrated that TNF-α-mediated signaling through TNFR2 triggers a signaling cascade that culminates in the activation of the nuclear transcription factor NFkB (Thommesen et al. “Distinct differences between TNF receptor 1- and TNF receptor 2-mediated activation of NFkappaB”. J Biochem Mol Biol. 2005 May 31;38(3):281-9; Yang et al. “Role of TNF-TNF Receptor 2 Signal in Regulatory T Cells and Its Therapeutic Implications”. Front Immunol. 2018 Apr 19;9:784). This, in turn, leads to cell activation and the synthesis of several proinflammatory factors, one of which is IFN-γ in NK cells (Liu et al. “NF-κB signaling in inflammation”. Signal Transduct Target Ther. 2017; 2. pii: 17023; Tato et al. “Opposing roles of NF-kappaB family members in the regulation of NK cell proliferation and production of IFN-gamma”. Int Immunol. 2006 Apr;18(4):505-13). In this paper, the terms TNFR2 signaling and TNFR2 activation are used interchangeably. Antibody molecules specifically bind to TNFR2. It is well known that an antibody specifically binds or interacts with a particular target molecule or antigen, and that this means that the antibody preferentially and selectively binds its target over a molecule that is not a target. By "an antibody molecule that specifically binds TNFR2" or "a TNFR2-specific antibody molecule" we mean an antibody that binds the TNFR2 protein in a dose-dependent manner but not to an unrelated protein. Furthermore, the same antibody binds cells that endogenously express TNFR2, and this binding can be blocked by pre-incubating the same cells with a commercially available polyclonal TNFR2 antibody reagent, demonstrating that non-specific binding cannot be detected when TNFR2 is masked by the polyclonal reagent. This is shown in Example 2.

An antibody molecule that specifically binds TNFR2 (or an anti-TNFR2 antibody molecule) refers to an antibody molecule that specifically binds to at least one epitope in the extracellular domain of TNFR2. Cell surface antigen and epitope are terms that can be easily understood by a person skilled in the field of immunology or cell biology.

Methods for assessing protein binding are known to those skilled in the art of biochemistry and immunology. It would be desirable for those skilled in the art to be able to use these methods to assess the binding of an antibody to a target and/or the binding of the Fc region of an antibody to an Fc receptor, and to assess the relative strength or specificity of, or inhibition or reduction of, or interference with these interactions. Examples of methods that can be used to assess protein binding include, for example, immunoassays, BIAcore, Western blotting, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FACS). See Fundamental Immunology, 2nd edition, Raven Press, New York, pp. 332-336 (1989) for a discussion of antibody specificity.

The target cells expressing TNFR2 to which the agonist antibody of the present invention binds may be any immune cells expressing TNFR2, such as CD8 positive cells and myeloid cells.

The effect of binding of the agonist antibody molecules of the invention to TNFR2 may be expressed in the activation of T cells and/or myeloid cells; and/or infiltration of T cells and/or myeloid cells into the affected tissue; and/or a change in the composition of T cells and/or myeloid cells in the affected tissue. A change in the composition of T cells and/or myeloid cells means herein different absolute or relative amounts of different cell subsets, such as Tregs, CD8-positive cells, tumor associated macrophages (TAMs) (including their different subsets), myeloid-derived suppressor cells (MDSCs) and/or proinflammatory macrophages.

In this context, “lesional tissue” means either tumor tissue (i.e., all cells in the tumor microenvironment, including tumor cells, immune cells, endothelial cells, and stromal cells) or tissue affected by a chronic inflammatory disease.

To determine whether an antibody molecule blocks - or rather, in the context of the present invention, does not block - the binding of a ligand to TNFR2, an ELISA assay can be used that determines the amount of TNF-α ligand bound to an immobilized TNFR2 receptor in the presence of antibodies specific for TNFR2. A non-blocking antibody does not interfere with the binding of TNF-α ligand to an immobilized TNFR2 receptor. This is demonstrated and explained in more detail in Example 3 below. More specifically, a non-blocking TNFR2 antibody molecule according to the present invention is an antibody molecule that reduces the binding of TNF-α to TNFR2 by less than 50% compared to the binding of TNF-α in the presence of an isotype control antibody molecule alone. In some embodiments of the present invention, this is determined in a high-dose single-point ELISA or a dose titration ELISA, as shown in Example 3 and in Fig. 6 and Fig. 7.

In contrast, a blocking antagonist antibody molecule is a complete blocker that is also capable of antagonizing TNFR2 signaling. Such antibody molecules are used for comparison in the examples below. A complete blocker is defined herein as an antibody molecule that reduces TNF-α binding to TNFR2 by greater than 98%, i.e., up to 100%, compared to TNF-α binding in the presence of an isotype control antibody molecule alone. An isotype control antibody is an antibody produced against a protein or other structure that is not present in any form in the assay being studied. An isotype control ideally has the same structure, but at least the same Fc portion, as the antibodies being compared. This is well known to one of skill in the art. In the examples described herein, the isotype control had the same backbone, the same Fc portion, and was specific for Fluorescein Isothiocyanate (FITC). In some embodiments of the invention, a complete blocker reduces TNF-α binding by greater than 99.5%. Other types of blockers are partial blockers and weak blockers. As used herein, a partial blocker is an antibody molecule that reduces TNF-α binding to TNFR2 by 60-98% compared to TNF-α binding in the presence of an isotype control antibody molecule alone, and a weak blocker is an antibody molecule that reduces TNF-α binding to TNFR2 by less than 60%, such as 50-59.9%, compared to TNF-α binding in the presence of an isotype control antibody molecule alone.

Full blocking antagonist antibody molecules, partial blocking antibody molecules and weak blocking antibody molecules are used in the examples for comparison with the agonist non-blocking antibody molecules of the present invention.

Several properties and features may underlie and (co-)determine the biological activity of antibodies. In addition to the ability to block or not block ligand-receptor binding, such important properties include the ability of antibody molecules to modulate receptor signaling, i.e., to agonize or antagonize receptor signaling, and the dependence of antibodies on FcγR interactions to confer therapeutic activity.

We first characterized the ability of blocking, partially blocking, and non-blocking antibodies to modulate TNFR2 signaling. Two extremes were identified.

In the first case, we identified antibodies that completely blocked ligand binding to TNFR2, which blocked TNF-α-induced TNFR2 signaling, and that did not themselves induce signaling when bound to cellular endogenously expressed TNFR2. This group of ligand-blocking, antagonist antibodies constitutes a separate invention and is included herein for comparison.

On the other hand, we have identified antibodies that do not block ligand binding to TNFR2, but when binding to TNFR2, endogenously expressing cells agonize the receptor. This second group of antibodies forms the basis of the present invention.

In this context, a non-blocking antibody is an antibody molecule that reduces the binding of TNF-α to TNFR2 by 0-50% (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%, including all whole numbers and all decimal numbers in between) compared to the binding of TNF-α in the presence of an isotype control antibody molecule alone.

Antibodies and categories defined as partial blocking agonist, partial blocking non-agonist, and full blocking non-antagonist were further identified, demonstrating the complex biology and great heterogeneity of anti-TNFR2 antibodies, clearly demonstrating that the antibodies of the present invention form a unique group.

To determine whether an antibody has agonist or antagonist activity, a natural killer (NK) cell assay can be used, as described in Example 4. Briefly, NK cells respond to IL-2 and IL-12 stimuli by secreting IFN-γ. Soluble TNF-α is produced endogenously and is present at consistent but suboptimal concentrations (~100 pg/mL) for TNFR2 signaling, meaning that IFN-γ can be either increased or decreased by modulating TNFR2 signaling. Therefore, exogenous addition of TNF-α at an optimal concentration for TNFR2 signaling increases IFN-γ concentrations in this assay, as does incubation with an agonist anti-TNFR2 antibody. In contrast, co-incubation with the anti-TNF-α antibody or the ligand blocking antagonist antibodies described herein for comparison reduced IFN-γ release in this assay. Thus, this assay can be used to identify the agonist or antagonist activity or lack thereof of anti-TNFR2 antibodies. (TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2. Almishri W. et al. J Innate Immun. 2016;8:617-629)

Therefore, the ability of antibodies to agonize, i.e., induce TNFR2 signaling, can be monitored using this assay. The ability of antibodies to induce signaling themselves upon binding to TNFR2 in the same natural killer (NK) cell assay can be assessed by monitoring and comparing the increase in IFN-γ release to that observed after culture in the presence or absence of exogenous TNF-α added at optimal signaling concentrations as described in Example 4. Therefore, a TNFR2 agonist antibody can be defined as an antibody that enhances IFN-γ release by NK cells in this assay. An antibody with intrinsic agonist capacity enhances IFN-γ release by NK cells in a manner that is independent of antibody cross-linking or interaction with Fc-gamma receptors, and is independent of the presence of soluble TNF-α ligand. Therefore, intrinsic agonist activity can be assessed using antibody formats that do not interact productively with FcγR, such as aglycosylated antibodies carrying the N297A mutation in the Fc domain, or in assay systems/cells lacking FcγR. NK cells are well known to those skilled in the art (Binyamin, L., et al (2008). Journal of immunology 180, 6392-6401; Blocking NK cell inhibitory self-recognition promotes antibody-dependent cellular cytotoxicity in a model of anti-lymphoma therapy.). Using this assay, an agonist antibody is defined as one that results in a > 100% (> 2-fold) increase in IFN-γ release. Since this assay uses primary cells from PBMC donors, at least 4 donors must be included and average values calculated for all donors. Cells from each donor to be included in the calculation of the mean must respond to positive control (soluble TNF-α) treatment with >100% (>2-fold) increase in IFN-γ levels compared to isotype control treatment.

The agonist antibody molecules described herein have intrinsic agonist activity as explained above.

In some embodiments of the invention, it is preferred that the antibody increases the release of IFN-γ by NK cells in the above assay by at least 100%.

In some embodiments of the invention, agonist activity may be improved by an antibody molecule that binds to an Fcγ receptor in addition to binding to TNFR2. In some such embodiments of the invention, agonist non-blocking TNFR2 antibody molecules bind with higher affinity to inhibitory Fcγ receptors than to activating Fcγ receptors. By having higher affinity for inhibitory Fcγ receptors than to activating Fcγ receptors, we include variant values that bind with higher affinity to inhibitory Fcγ receptors compared to individual activating Fcγ receptors, for example, compared to any of FcγRIIA, FcγRIIIA, and FcγRI.

The relatively high homology between the mouse and human FcγR systems accounts for many common aspects of conserved FcγR-mediated mechanisms between the species. However, mouse and human IgG subclasses differ in their affinity for cognate FcγRs, an important consideration when translating FcγR-mediated observations in the mouse system to human IgG-based therapeutics is to select an antibody, antibody subclass, and/or engineered subclass variant that exhibit appropriate binding to human activating versus inhibitory FcγRs. The affinity and/or avidity of human antibody molecules for individual human FcγRs can be determined using surface plasmon resonance (SPR).

In some embodiments of the invention, binding to the Fc receptor occurs through a normal interaction between the Fc region of the agonist antibody molecule and the Fc receptor. In some such embodiments of the invention, the antibody molecule is an IgG that has an Fc region that binds to an Fcγ receptor. In some such embodiments of the invention, the anti-TNFRII antibody is a human IgG2 isotype that has similar intermediate affinity for human inhibitory FcγRIIB and human activating FcγRIIA and FcγRIIIA, but does not interact productively with human activating FcγRI. In some embodiments of the present invention, the anti-TNFRII antibody is of the human IgG1 isotype, which binds FcγRIIB with higher affinity than IgG2, but also binds activating human FcγRIIA, FcγRIIIA with higher affinity and additionally binds activating FcγRI with high affinity. In other embodiments of the invention, the anti-TNFRII antibody is a human IgG engineered to have enhanced binding to FcγRIIB, such as the “SELF” mutation (Chu et al. “Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies.” Mol Immunol. 2008 Sep; 45 (15): 3926-33) and/or engineered to have relatively increased binding to FcγRIIB compared to activation of FcγR, such as the V9 or V11 mutations (Mimoto et al. “Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa ^R131 and FcγRIIa ^H131 ”. Protein Eng Des Sel. 2013 Oct; 26 (10): 589–598). Such IgG variants engineered for enhanced binding to inhibitory FcγRIIB or specifically increased binding affinity to inhibitory FcγRIIB but not activating FcγRIIA have been shown to enhance agonist activity in vivo and the therapeutic activity of the CD40 agonist antibody CP-870893 in humanized animals for FcγR activation and inhibition (Dahan et al. 2016. 'Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcgammaR Engagement', Cancer Cell, 29: 820–31).

The Fc receptor, to which the agonist antibody molecule can bind in addition to TNFR2, is a receptor found on the surface of cells of myeloid origin such as macrophages, monocytes, MDCS, neutrophils, mast cells, basophils or dendritic cells, or on the surface of lymphocytes such as NK cells, B cells or certain T cells.

As mentioned above, antibody molecules often bind to Fc receptors through their Fc regions. Because the agonist antibody molecules described herein have intrinsic agonist activity, they do not need to bind to Fc receptors to agonize TNFR2. This means that in some embodiments of the invention, antibody molecules can be used that are not dependent on Fc receptor binding through its Fc region, and in fact, antibody molecules that do not have an Fc region can be used. In some such embodiments of the invention, the antibody molecule can be a Fab' ₂ or a pegylated version thereof. In some embodiments of the invention, the antibody molecules can be a bivalent or multivalent antibody molecule comprising single chain antibodies, Fab, Fv, scFv, Fab and/or (Fab') ₂ . In other embodiments of the invention, the antibody molecules may comprise a modified Fc region, such as an aglycosylated version of an IgG1 antibody molecule. Such aglycosylation can be achieved, for example, by substituting an amino acid, asparagine, at position 297 (N297X) in the antibody chain. The substitution can be glutamine (N297Q) or alanine (N297A) or glycine (N297G) or asparagine (N297D) or serine (N297S). Other substitutions are, for example, described by Jacobsen FW et al., JBC 2017, 292, 1865-1875 (see, for example, Table 1); Such additional replacements include L242C, V259C, A287C, R292C, V302C, L306C, V323C, I332C and/or K334C.

In some embodiments of the present invention, the TNFR2 agonist antibody molecule is an IgG1, IgG3, or IgG4 antibody molecule.

In some embodiments of the invention, the TNFR2 agonist antibody molecule is an IgG antibody molecule that exhibits improved binding to one or more activating Fc receptors and/or is engineered to improve binding to one or more activating Fcγ receptors and/or is engineered to improve the relative binding to activating receptors over inhibitory Fcγ receptors. In some embodiments of the invention, the anti-TNFR2 antibody is an Fc-engineered human IgG1 antibody. Examples of such engineered antibody variants include afucosylated antibodies with selectively improved antibody binding to FcγRIIIA and antibodies engineered by targeted, mutational, or other amino acid substitution resulting in improved binding to one or more activating Fcγ receptors relative to inhibitory FcγRIIB. (Richards et al. 2008. 'Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells', Mol Cancer Ther, 7: 2517-27; Lazar et al. 2006. 'Engineered antibody Fc variants with enhanced effector function', Proc Natl Acad Sci U S A, 103: 4005-10).

In some embodiments of the invention, a human IgG antibody that is engineered to improve binding to activating Fc gamma receptors may be a human IgG antibody that has two mutations S239D and I332E or three mutations S239D, I332E and A330L, and/or a G236A mutation in its Fc region. In some embodiments of the invention, a human IgG antibody that is engineered to improve binding to activating Fc gamma receptors may be an afucosylated human IgG antibody.

As explained above, the antibody molecules being intrinsic agonists means that they are agonists in both the absence and presence of TNF-α. In some embodiments of the invention, the antibody is agonistic in the absence of TNF-α. In some embodiments of the invention, the antibody is agonistic in the presence of TNF-α.

The target cell expressing TNFR2 to which the agonist antibody binds according to the present invention may be selected from the group consisting of immune cells expressing TNFR2 or cancer cells.

Antibodies are well known to those skilled in the fields of immunology and molecular biology. Typically, an antibody consists of two heavy (H) chains and two light (L) chains. This complete antibody molecule is sometimes referred to herein as a full-length antibody or full-length antibody. The heavy chain of an antibody contains one variable domain (VH) and three constant domains (CH1, CH2, and CH3), and the light chain of an antibody molecule contains one variable domain (VL) and one constant domain (CL). The variable domains (sometimes collectively referred to as the F _V region) bind to the antibody target, or antigen. Each variable domain consists of three loops, called complementarity determining regions (CDRs), that are responsible for target binding. The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions. Antibodies or immunoglobulins can be classified into different classes depending on the amino acid sequence of the constant region of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and in humans, some of these immunoglobulins are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2.

Another part of the antibody is the Fc region (also known as the crystallizable fragment domain), which consists of two constant domains from each of the antibody's heavy chains. As mentioned above, the Fc region is responsible for the interaction between the antibody and the Fc receptor.

The term "antibody molecule" as used in this application refers to full-length or full-length antibodies, as well as functional fragments of full-length antibodies and derivatives of such antibody molecules.

Functional fragments of a full-length antibody have the same antigen binding characteristics as the corresponding full-length antibody and include either the same variable domains (i.e., VH and VL sequences) and/or the same CDR sequences as the corresponding full-length antibody. A functional fragment does not always contain all six CDRs of the corresponding full-length antibody. It should be noted that molecules containing three or fewer CDR regions (in some cases even only one CDR region or part of it) are able to retain the antigen-binding activity of the antibody from which the CDRs are derived. For example, Gao et al., 1994, J. Biol. Chem., 269: 32389-93, reported that the entire VL chain (including all three CDR regions) has high affinity for its substrate.

Molecules containing two CDR regions are described, for example, in Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Proposal Screening, 4: 417–430. On page 418 (right column – 3 “Our design strategy”) a minibody is described that includes only the hypervariable regions of the H1 and H2 CDRs interspersed within framework regions. This minibody is described as being able to bind to the target. Pessi et al., 1993, Nature, 362: 367–9, and Bianchi et al., 1994, J. Mol. Biol., 236: 649–59, are cited by Vaughan and Sollazzo and describe the H1 and H2 minibodies and their properties in more detail. Qiu et al., 2007, Nature Biotechnology, 25:921-9, show that a molecule consisting of two linked CDR regions is capable of binding antigen. Quiocho 1993, Nature, 362: 293-4, provides a brief description of the "minibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7, comments that molecules containing two CDR regions are able to retain antigen-binding activity.

Single CDR antibody molecules are described, for example, by Laune et al., 1997, JBC, 272: 30937–44, who showed that a number of CDR-derived hexapeptides exhibited antigen-binding activity and noted that synthetic peptides of the complete single CDR exhibited strong binding activity. Monnet et al., 1999, JBC, 274: 3789–96, showed that a number of 12-mer peptides and their associated framework regions possessed antigen-binding activity and commented that the CDR3-like peptide alone was capable of binding antigen. Heap et al., 2005, J. Gen. Virol., 86: 1791–1800, reported that a “microantibody” (a molecule containing a single CDR region) was able to bind antigen, and a cyclic peptide from an anti-HIV antibody was shown to have antigen-binding activity and function. Nicaise et al., 2004, Protein Science, 13: 1882–91, showed that a single CDR region can confer antigen-binding activity and affinity for its lysozyme antigen.

Thus, antibody molecules having five, four, three or fewer CDR regions are able to retain the antigen-binding properties of the full-length antibodies of which they are derivatives.

Such an antibody molecule may also be a derivative of a full-length antibody or a fragment of such an antibody. If a derivative is used, it must have the same antigen-binding characteristics as the corresponding full-length antibody, in the sense that it binds to the same epitope on the target as the full-length antibody.

Thus, the term "antibody molecule" as used in the present application refers to all types of antibody molecules and their functional fragments, as well as their derivatives, including: monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, antibodies of human origin, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), Fab fragments, F(ab') ₂ fragments, F(ab') fragments, Fvs (sdFv) with a disulfide bond, antibody heavy chains, antibody light chains, antibody heavy chain homodimers, antibody light chain homodimers, antibody heavy chain heterodimers, antibody light chain heterodimers, antigen-binding functional fragments of such homo- and heterodimers.

Additionally, the term "antibody molecule" as used in this application includes all classes of antibody molecules and functional fragments, including: IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD and IgE, unless otherwise specified.

In some embodiments of the invention, the antibody molecule is a human antibody molecule, a humanized antibody molecule, or an antibody molecule of human origin. In some such embodiments of the invention, the antibody molecule is an IgG antibody. It is known that optimal co-stimulation of TNFR superfamily agonist receptors, such as TNFR2, depends on the interaction of the antibodies with inhibitory FcγRII. In mice, the IgG1 isotype, which binds preferentially to the inhibitory Fc gamma receptor (FcγRIIB) and only weakly to activating Fc gamma receptors, is known to be optimal for the co-stimulatory activity of monoclonal antibodies directed to the TNFR superfamily. Although a direct human equivalent of the mouse IgG1 isotype has not been described, antibodies can be engineered to exhibit similar enhancement of binding to inhibitory, rather than activating, human Fc gamma receptors. Such engineered TNFR superfamily-targeted antibodies also exhibit improved in vivo costimulatory activity in transgenic mice engineered to express activating and inhibitory human Fc gamma receptors (Dahan et al, 2016, Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement. Cancer Cell. 29(6):820-31). Thus, in some embodiments of the invention, the antibody molecule has an isotype that optimally engages inhibitory Fc receptors. In some embodiments of the invention, the antibody molecule is an IgG2 antibody.

In some embodiments of the present invention, an agonist antibody molecule that specifically binds to TNFR2 may be a llama antibody, and in particular a llama hcIgG antibody. Like all mammals, camelids produce conventional antibodies consisting of two heavy chains and two light chains linked together by disulfide bonds in a Y shape (IgG ₁ ). However, they also produce two unique subclasses of immunoglobulin G, IgG ₂ and IgG ₃ , also known as IgG heavy chains (hcIgG). These antibodies consist of only two heavy chains that lack a CH1 region, but still have an antigen-binding domain at their N-terminus, called V _and H. Conventional Ig requires the association of variable regions from both the heavy and light chains to provide a wide variety of antigen-antibody interactions. Although isolated heavy and light chains do exhibit this ability, they exhibit very low affinity compared to paired heavy and light chains. A unique feature of hcIgG is the ability of their monomeric antigen-binding regions to bind antigens with a specificity, affinity, and especially diversity comparable to conventional antibodies, without the need for association with another region.

As indicated above, the present invention includes various types and forms of antibody molecules, which should be known to a person skilled in the art of immunology. It is well known that antibodies used for therapeutic purposes are often modified by additional components that change the properties of the antibody molecule.

Accordingly, we mean that an antibody molecule described herein or an antibody molecule used as described herein (e.g., a monoclonal antibody molecule and/or a polyclonal antibody molecule and/or a bispecific antibody molecule) comprises a detectable moiety and/or a cytotoxic moiety.

By "detectable molecular fragment" we mean one or more groups consisting of: an enzyme; a radioactive atom; a fluorescent molecular fragment; a chemiluminescent molecular fragment; a bioluminescent molecular fragment. The detectable molecular fragment allows the antibody molecule to be visualized in vitro and/or in vivo and/or ex vivo.

By "cytotoxic moiety" we mean a radioactive moiety and/or an enzyme, for example, where the enzyme is a caspase; and/or a toxin, for example, where the toxin is a bacterial toxin or poison; where the cytotoxic moiety is capable of inducing cell lysis.

It is also contemplated in this invention that the antibody molecule may be in isolated form and/or purified form, and/or may be PEGylated. PEGylation is a method by which polyethylene glycol polymers are added to a molecule, such as an antibody molecule or derivative, in order to change its behavior, such as to extend its half-life by increasing its hydrodynamic size, preventing renal excretion.

As discussed above, the CDRs of an antibody bind to a target antibody. The amino acid assignments to each CDR region described in this application correspond to the definitions in Kabat EA et al. 1991, in "Sequences of Proteins of Immunological Interest," fifth edition, NIH Publication No. 91-3242, pp xv-xvii.

As may be known to one skilled in the art, there are other ways of assigning amino acids to each CDR region. For example, the International Immunogenetics Information System (IMGT(R)) (http://www.imgt.org/ and Lefranc and Lefranc "The Immunoglobulin FactsBook" published by Academic Press, 2001).

In some embodiments of the present invention, the antibody molecule that specifically binds TNFR2 is a human antibody.

In some embodiments of the invention, an antibody molecule that specifically binds TNFR2 is an antibody of human origin, i.e., an originally human antibody that has been modified as described herein.

In some embodiments of the invention, an antibody molecule that specifically binds TNFR2 is a humanized antibody, i.e., an originally non-human antibody that has been modified to increase its similarity to a human antibody. Humanized antibodies can be, for example, mouse antibodies or llama antibodies.

In some embodiments of the present invention, the antibody molecule that specifically binds TNFR2 is a human IgG2 antibody molecule.

In some embodiments of the invention, the anti-TNFR2 antibody is an antibody in the form of a human IgG2 antibody that exhibits improved binding to one or more inhibitory Fc receptors and/or is engineered to improve binding to one or more inhibitory Fc receptors; accordingly, in some embodiments of the invention, the anti-TNFR2 antibody is an Fc-engineered human IgG2 antibody.

In some embodiments of the invention, the anti-TNFR2 antibody is a murine or humanized murine IgG3 antibody.

In some embodiments of the invention, the anti-TNFR2 antibody is a monoclonal antibody.

In some embodiments of the invention, the anti-TNFR2 antibody is a polyclonal antibody.

In some embodiments of the present invention, an antibody molecule that specifically binds TNFR2 is a human IgG1 antibody molecule that corresponds to or murine IgG2a.

In some embodiments of the present invention, an antibody molecule that specifically binds TNFR2 comprises one of the VH-CDR1 sequences listed in Table 1 below.

In some embodiments of the present invention, an antibody molecule that specifically binds TNFR2 comprises one of the VH-CDR2 sequences listed in Table 1 below.

In some embodiments of the present invention, an antibody molecule that specifically binds TNFR2 comprises one of the VH-CDR3 sequences listed in Table 1 below.

In some embodiments of the present invention, an antibody molecule that specifically binds TNFR2 comprises one of the VL-CDR1 sequences listed in Table 1 below.

In some embodiments of the present invention, an antibody molecule that specifically binds TNFR2 comprises one of the VL-CDR2 sequences listed in Table 1 below.

In some embodiments of the present invention, an antibody molecule that specifically binds TNFR2 comprises one of the VL-CDR3 sequences listed in Table 1 below.

In some embodiments of the invention, the anti-TNFR2 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules comprising 6 CDRs selected from the group consisting of:

SEQ ID NO: 1, 2, 3, 4, 5 and 6;

SEQ ID NO: 9, 10, 11, 12, 13 and 14;

SEQ ID NO: 17, 18, 19, 20, 21 and 22;

SEQ ID NO: 25, 26, 27, 28, 29 and 30;

SEQ ID NO: 33, 34, 35, 36, 37 and 38;

SEQ ID NO: 41, 42, 43, 44, 45 and 46;

SEQ ID NO: 49, 50, 51, 52, 53 and 54;

SEQ ID NO: 57, 58, 59, 60, 61 and 62;

SEQ ID NO: 65, 66, 67, 68, 69 and 70;

SEQ ID NO: 73, 74, 75, 76, 77 and 78;

SEQ ID NO: 81, 82, 83, 84, 85 and 86;

SEQ ID NO: 89, 90, 91, 92, 93 and 94; and

SEQ ID NO: 97, 98, 99, 100, 101 and 102.

In some embodiments of the invention, the anti-TNFR2 antibody molecule is an antibody molecule comprising 6 CDRs having the sequences of SEQ ID NOs: 1, 2, 3, 4, 5 and 6; or an antibody molecule comprising 6 CDRs having SEQ ID NOs: 9, 10, 11, 12, 13 and 14; or an antibody molecule comprising 6 CDRs having SEQ ID NOs: 17, 18, 19, 20, 21 and 22; or an antibody molecule comprising 6 CDRs having SEQ ID NOs: 25, 26, 27, 28, 29 and 30; or an antibody molecule comprising 6 CDRs having SEQ ID NOs: 33, 34, 35, 36, 37 and 38; or an antibody molecule comprising 6 CDRs having SEQ ID NO: 41, 42, 43, 44, 45 and 46.

In some embodiments of the invention, the anti-TNFR2 antibody molecule is an antibody molecule comprising 6 CDRs having the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

In some embodiments of the invention, the anti-TNFR2 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules comprising a VH selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, and 103.

In some embodiments of the invention, the anti-TNFR2 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules comprising a VL selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, and 104.

In some embodiments of the invention, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VH of SEQ ID NO: 7, 15, 23, 31, 39, or 47.

In some embodiments of the invention, the anti-TNFR2 antibody molecule is an antibody molecule comprising a VH of SEQ ID NO: 7.

In some embodiments of the invention, it is preferred that the anti-TNFR2 antibody molecule is an antibody molecule comprising a VL having SEQ ID NO: 8, 16, 24, 32, 40 or 48.

In some embodiments of the invention, it is more preferred that the anti-TNFR2 antibody molecule is an antibody molecule comprising a VL having SEQ ID NO: 8.

In some embodiments of the present invention, it is preferred that the anti-TNFR2 antibody molecule comprises a VH having SEQ ID NO: 7 and a VH having SEQ ID NO: 8.

In some embodiments of the invention, the anti-TNFR2 antibody molecule comprises a CH having SEQ ID NO: 217.

In some embodiments of the invention, the anti-TNFR2 antibody molecule comprises a CL having SEQ ID NO: 218.

In some embodiments of the invention, an anti-TNFR2 antibody molecule comprises a VH having SEQ ID NO: 7, a VH having SEQ ID NO: 8, a CH having SEQ ID NO: 217, and a CL having SEQ ID NO: 218.

Table 1. Specific sequences of TNFR2 agonist antibody molecules that do not block TNF-α binding to TNFR2 as described herein (in VH and VL sequences, CDR sequences in bold)

Antibody clone Plot Subsequence SEQ ID NO: 001-F02 VH-CDR1 FSDYYMSWVRQAPG 1 VH-CDR2 ANINTDGSEKYYLDSVKGR 2 VH-CDR3 AREEYGAFDI 3 VL-CDR1 CSGSSSNIGSNTVN 4 VL-CDR2 DNNKRPS 5 VL-CDR3 CQSFDRGLSGSIV 6 VH 7 VL 8 001-F06 VH-CDR1 FSSYAMHWVRQAPG 9 VH-CDR2 SAISGGATTTYYADSVKGR 10 VH-CDR3 AKGGTGDPYYFDY 11 VL-CDR1 CTGSSSNIGAGYDVH 12 VL-CDR2 RNNQRPS 13 VL-CDR3 CAARDDGLSGPV 14 VH 15 VL 16 001-B05 VH-CDR1 FSNAWMSWVRQAPG 17 VH-CDR2 SSISSASGYIYYGDSVKGR 18 VH-CDR3 ARGTLYGDFDEF 19 VL-CDR1 CSGSSSNIGNNAVN 20 VL-CDR2 GNTNRPS 21 VL-CDR3 CQSYDSSLSGYVV 22 VH 23 VL 24 001-C05 VH-CDR1 FSSNEMSWIRQAPG 25 VH-CDR2 SVIYSGGSTYYADSVKGR 26 VH-CDR3 ARREGWLVPFDY 27 VL-CDR1 CSGSSSNIGSNTVN 28 VL-CDR2 GNIIRPS 29 VL-CDR3 CQSFDTTLSGSIV 30 VH 31 VL 32 004-E08 VH-CDR1 FSRYWMHWVRQVPG 33 VH-CDR2 SGISDSGVVTYYADSVKGR 34 VH-CDR3 ARAQSVAFDI 35 VL-CDR1 CSGSSSNIGAGHDVH 36 VL-CDR2 YDDLLPS 37 VL-CDR3 CAAWDDSLSGWV 38 VH 39 VL 40 001-G05 VH-CDR1 FSSYAMSWVRQAPG 41 VH-CDR2 SVISGSGGSTYYADAVKGR 42 VH-CDR3 TTDSGSGSIL 43 VL-CDR1 CTGSSSNIGAGYDVH 44 VL-CDR2 SNNQRPS 45 VL-CDR3 CAAWDDSLNGPV 46 VH 47 VL 48 001-A09 VH-CDR1 FSSNYMSWVRQAPG 49 VH-CDR2 SVISGSGGSTYYADSVKGR 50 VH-CDR3 ARDRGWFDP 51 VL-CDR1 CSGSRSNIDNSYVS 52 VL-CDR2 RNNQRPS 53 VL-CDR3 CATWDDSLSGPV 54 VH 55 VL 56 001-B09 VH-CDR1 FSRHAMNWVRQAPG 57 VH-CDR2 SSISTGSSYIDYADSVKGR 58 VH-CDR3 AREKGHYYYGMDV 59 VL-CDR1 CTGSSSNIGAGYDVH 60 VL-CDR2 GNSYRPS 61 VL-CDR3 CQSYDTSLSAYVV 62 VH 63 VL 64 001-C03 VH-CDR1 FSNAWMSWVRQAPG 64 VH-CDR2 SAISVSGINTYYADSVKGR 66 VH-CDR3 ARDTGSLGVDY 67 VL-CDR1 CSGSSSNIGSNTVN 68 VL-CDR2 RNNQRPS 69 VL-CDR3 CQSYDSSLSISV 70 VH 71 VL 72 001-A10 VH-CDR1 FSDYYMTWIRQAPG 73 VH-CDR2 SSISGGSTYYADSRKGR 74 VH-CDR3 AREPGYSYGFFDY 75 VL-CDR1 CTGSSSNIGAGYDVH 76 VL-CDR2 SNNQRPS 77 VL-CDR3 CQSYDRSLSGSIV 78 VH 79 VL 80 001-C06 VH-CDR1 SSSYWMSWVRQAPG 81 VH-CDR2 SAISGSGGSTYYADSVKGR 82 VH-CDR3 AREYSGYEFDF 83 VL-CDR1 CTGSSSNIGARSDVH 84 VL-CDR2 GNRNRPS 85 VL-CDR3 CQSFDRGLSGSIV 86 VH 87 VL 88 001-H03 VH-CDR1 FSSNYMSWVRQAPG 89 VH-CDR2 SSISSSSSSYIYYADSVKGR 90 VH-CDR3 ARDRGRTGTDY 91 VL-CDR1 CSGTTSNIGSYAVN 92 VL-CDR2 GNINRPS 93 VL-CDR3 CQSYDSSLSASL 94 VH 95 VL 96 005-A05 VH-CDR1 FSSYAMSWVRQAPG 97 VH-CDR2 STIIGSGANTWYADSVKGR 98 VH-CDR3 ARHEGYYYYGMDV 99 VL-CDR1 CTGSSSNIGAGYVVH 100 VL-CDR2 GNSNRPS 101 VL-CDR3 CAAWDDSLNGRV 102 VH 103 VL 104

To determine or demonstrate the properties of the antibody molecules of the present invention, they were compared with antibody molecules that block TNF-α from binding to TNFR2. Such antibodies are shown in Table 2.

Table 2. Specific sequences of TNFR2 blocking antibody molecules mentioned in this document as reference antibodies (in VH and VL sequences, CDR sequences are in bold)

Antibody clone Plot Subsequence SEQ ID NO: 001-H10 VH-CDR1 FDDYGMSWVRQAPG 105 VH-CDR2 SVIYSGGSTYYADSVKGR 106 VH-CDR3 CARDRSSSWYRDGMDV 107 VL-CDR1 CTGSSSNIGAGYDVH 108 VL-CDR2 GNSNRPS 109 VL-CDR3 CAAWDDSLSGWV 110 VH 111 VL 112 004-H02 VH-CDR1 FDDYGMSWVRQAPG 113 VH-CDR2 STIYSGDNAYYGASVRGR 114 VH-CDR3 ARVYSSWRKRAFDI 115 VL-CDR1 CSGTSSNIESNTVN 116 VL-CDR2 SDNQRPS 117 VL-CDR3 CAAWDDSLSGWV 118 VH 119 VL 120 005-B02 VH-CDR1 FSDYYMSWIRQAPG 121 VH-CDR2 ALIWYDGGNEYYADSVKGR 122 VH-CDR3 VRETGNYGMDV 123 VL-CDR1 CTGSSSNIGAGYDVH 124 VL-CDR2 RNNQRPS 125 VL-CDR3 CATWDDRVNGPV 126 VH 127 VL 128 005-B08 VH-CDR1 FSDYYMSWIRQAPG 129 VH-CDR2 AIISYDGGGKYFADPVKGR 130 VH-CDR3 ARYYGDGGFDP 131 VL-CDR1 CTGSSSNIGAGYVVH 132 VL-CDR2 SNNQRPS 133 VL-CDR3 CAAWDDSLNGPV 134 VH 135 VL 136 001-E06 VH-CDR1 FSSNYMSWVRQAPG 137 VH-CDR2 ALIWYDGSNKYYADSVKGR 138 VH-CDR3 AKDPLFDS 139 VL-CDR1 CTGRSSNIGAGYDVH 140 VL-CDR2 DNNKRPS 141 VL-CDR3 CAAWDDSLNGPV 142 VH 143 VL 144 001-G04 VH-CDR1 FNTYSMNWVRQAPG 145 VH-CDR2 SVLYSDDDTHYADSVKGR 146 VH-CDR3 ARDCGGDCHSGDDAFDI 147 VL-CDR1 CSGSSSNIGSNTVN 148 VL-CDR2 DNDKRPS 149 VL-CDR3 CAAWHDSLNGWV 150 VH 151 VL 152 001-G10 VH-CDR1 FSAYGMHWVRQAPG 153 VH-CDR2 AVVSYDGREKHYADSVKGR 154 VH-CDR3 ARSDGGYDSDSGYY 155 VL-CDR1 CSGSTSNIGSNFVY 156 VL-CDR2 DNNKRPS 157 VL-CDR3 CSSYAYSDNIL 158 VH 159 VL 160 001-C08 VH-CDR1 FSNAWMSWVRQAPG 161 VH-CDR2 SGISSSGSSAYYADSVKGR 162 VH-CDR3 ARHYYYHIAGYYYDTFDI 163 VL-CDR1 CSGSSSNIGGNTVN 164 VL-CDR2 GNTNRPS 165 VL-CDR3 CAAWDDSLSGVV 166 VH 167 VL 168 001-H09 VH-CDR1 FSSYAMSWVRQAPG 169 VH-CDR2 ATISYHGSDKDYADSVKGR 170 VH-CDR3 ARDANYHSSGYYYDVFDI 171 VL-CDR1 CSGSSSNIGSNTVN 172 VL-CDR2 GNSNRPS 173 VL-CDR3 CAAWDDSLSTWV 174 VH 175 VL 176 005-F10 VH-CDR1 FSDYYMTWIRQAPG 177 VH-CDR2 SGISGSGGYIHYADSVKGR 178 VH-CDR3 AREGLLPDAFD 179 VL-CDR1 CSGSSSNIGNNYVS 180 VL-CDR2 RNNQRPS 181 VL-CDR3 CAAWDDSVSGWV 182 VH 183 VL 184 001-B11 VH-CDR1 185 VH-CDR2 186 VH-CDR3 187 VL-CDR1 188 VL-CDR2 SNNQRPS 189 VL-CDR3 190 VH 191 VL 192 001-C07 VH-CDR1 FSSYEMNWVRQAPG 193 VH-CDR2 STITGGGSIYDANSVQGR 194 VH-CDR3 ARDSTYHSSGYYYDVFDI 195 VL-CDR1 CSGSSSNIGSNTVN 196 VL-CDR2 GNSNRPS 197 VL-CDR3 CAAWDDSLSGHWV 198 VH 199 VL 200 001-D01 VH-CDR1 FSSYGMHWVRQAPG 201 VH-CDR2 SAVFGSGHGNTFYADAVKGR 202 VH-CDR3 AREQLWFGQDAFDI 203 VL-CDR1 CSGSSSNIGSNTVN 204 VL-CDR2 GNSNRPS 205 VL-CDR3 CQSYDSSLSASV 206 VH 207 VL 208 003-F10 VH-CDR1 FSDAWMTWVRQAPG 209 VH-CDR2 SDLSDSGGSTYYADSVKGR 210 VH-CDR3 GRLAAGGPVDY 211 VL-CDR1 CTGSSSNIGAGYDVH 212 VL-CDR2 SNNQRPS 213 VL-CDR3 CSVWDDSLNSWV 214 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSDAWMTWVRQAPGKGLEWVSDLSDSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGRLAAGGPVDYWGQGTLVTVSS 215 VL QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSVWDDSLNSWVFGGGTKLTVLG 216

All sequences in Tables 1 and 2 above are of human origin and come from the n-CoDeR® library, as explained in detail in Example 1.

In some embodiments of the invention, the antibody molecules that specifically bind TNFR2 described herein may also comprise one or both of the constant regions (CH and/or CL) listed in Table 3 below.

Table 3

Plot Subsequence SEQ ID NO: CH ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 217 CL QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 218 CH AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 219 CH AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 220 CL QPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS 221

The first CH (SEQ ID NO: 217) and the first CL (SEQ ID NO: 218) in Table 3 above are of human origin. The second CH (SEQ ID NO: 219) and the third CH (SEQ ID NO: 220) in Table 3 are both derived from mouse IgG2a, with the difference that the third CH sequence (SEQ ID NO: 220) contains the N297A mutation. The second CL sequence (SEQ ID NO: 221) is derived from the constant region of the mouse lambda light chain. These mouse sequences are used in the examples for surrogate antibodies.

In some embodiments of the invention, the antibody molecule does not bind human TNFR2 (hTNFR2). In some embodiments of the invention, it is preferred that the agonist antibody molecules bind tightly to human TNFR2, i.e., that they have a low EC50 value. This is demonstrated further in Example 2.

In some embodiments of the invention, it is preferred that the antibody molecule binds to both hTNFR2 and cynomolgus macaque TNFR2 (cmTNFR2 or cynoTNFR2). Cross-reactivity with TNFR2 expressed on cynomolgus macaque cells, also called crab-eating macaque or Macaca fascicularis, may be advantageous since it allows the antibody molecule to be tested in animals without the need to use a surrogate antibody, with particular attention to tolerability.

In some embodiments of the invention, it is necessary to use a surrogate antibody to test the functional activity of the antibody molecule in appropriate in vivo mouse models. To ensure comparability between the effect of the antibody molecule in humans and the in vivo results for the surrogate antibody in mice, it is important to select a functionally equivalent surrogate antibody that has the same in vitro characteristics as the human antibody molecule. In some embodiments of the invention, the antibody molecule does not specifically bind to an epitope of TNFR2 that contains or consists of a KCSPG sequence.

In some embodiments of the invention, an antibody molecule of the invention or used according to the invention is an antibody molecule that is capable of competing with the specific antibodies provided herein, such as capable of competing with antibody molecules comprising a VH selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95 and 103; and/or a VL selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96 and 104, for binding to TNFR2.

By "capable of competing for" is meant that the competing antibody is capable of inhibiting or otherwise interfering, at least in part, with the binding of an antibody molecule, as defined herein, to a particular TNFR2 target.

For example, such a competing antibody molecule may be capable of inhibiting the binding of an antibody molecule described herein to TNRF2 by at least about 10%; such as at least about 20%, or at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100%.

Competitive binding can be determined by methods well known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA).

ELISA assays can be used to evaluate epitope-modifying or blocking antibodies. Additional methods suitable for detecting competing antibody are described in Antibodies: A Laboratory Manual, Harlow & Lane, which is incorporated herein by reference (e.g., see pp. 567-569, 574-576, 583, and 590-612, 1988, CSHL, NY, ISBN 0-87969-314-2).

In some embodiments of the present invention, it is of interest to use not the antibody molecule itself, but a nucleotide sequence encoding such an antibody molecule. Thus, the present invention encompasses nucleotide sequences encoding the above-mentioned agonist non-blocking TNFR-2 antibody molecules.

The above-described agonist non-blocking antibody molecules and nucleotide sequences can be used in medicine, and then such antibody molecule and/or nucleotide sequence can be included in a pharmaceutical composition, as further discussed below.

The above-described agonist non-blocking antibody molecules, nucleotide sequences and/or pharmaceutical compositions can be used in the treatment of cancer, as further discussed below.

The above-described agonist non-blocking antibody molecules, nucleotide sequences and/or pharmaceutical compositions can be used to treat a chronic inflammatory disease, as further discussed below.

The above-described agonist non-blocking antibody molecules and/or nucleotide sequences can be used in the manufacture of a pharmaceutical composition for the treatment of cancer.

The above-described agonist non-blocking antibody molecules and/or nucleotide sequences can be used in the manufacture of a pharmaceutical composition for the treatment of a chronic inflammatory disease.

The above-described agonist non-blocking antibody molecules and/or pharmaceutical compositions can be used in a method of treating cancer in a patient, in which a therapeutically effective amount of the antibody molecule or pharmaceutical composition is administered to the subject.

The above-described agonist non-blocking antibody molecules and/or pharmaceutical compositions can be used in a method of treating a chronic inflammatory disease in a patient, in which a therapeutically effective amount of an antibody molecule or pharmaceutical composition is administered to the patient.

In some embodiments of the invention relating to the treatment of cancer, the cancer is a solid cancer or leukemic cancer. A solid tumor is an abnormal mass of tissue that does not typically contain cysts or fluid areas. Solid tumors can be benign (not cancer) or malignant (cancer). Malignant solid tumors are referred to herein as solid cancer. The various types of solid tumors or cancers are named after the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.

More specific examples of solid cancers include lung cancer, head and neck cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, bladder cancer, ovarian cancer, endometrial cancer, kidney cancer, liver cancer, pancreatic cancer, thyroid cancer, brain cancer, central nervous system cancer, melanoma, neuroblastoma, lymphoma, Wilms' tumor, rhabdomyosarcoma, retinoblastoma, and bone cancer.

More specific examples of leukemic cancer include acute lymphocytic leukemia, chronic myeloproliferative disorder, acute non-lymphocytic leukemia, acute B-cell lymphocytic leukemia, chronic lymphocytic leukemia, T-cell acute lymphocytic leukemia, non-Hodgkin's lymphoproliferative disorders. In some embodiments of the invention, the agonist non-blocking antibody molecules described above can be used in combination with an antibody molecule that specifically binds a checkpoint inhibitor. Alternatively, the nucleotide sequences encoding an agonist non-blocking TNFR2 antibody molecule discussed above can be used in combination with an antibody molecule that specifically binds a checkpoint inhibitor or a co-stimulatory agonist antibody. Examples of antibodies to checkpoint inhibitors include antibodies targeting CTLA4, PD1, PD-L1, VISTA, TIGIT, CD200, CD200R, BTLA, LAG3, TIM3, B7-H3, B7-H4, B7-H7. Examples of co-stimulatory agonist antibodies include antibodies targeting OX40, 41BB, OX40L, 41BBL, GITR, ICOS, DR3, DR4, DR5, CD40, CD27, RANK, HVEM, LIGHT, and B7-H6. Alternatively, the above-described agonist non-blocking TNFR2 antibody molecules can be used in combination with a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor or co-stimulatory agonist. Alternatively, the nucleotide sequences discussed above encoding an agonist non-blocking TNFR2 antibody molecule can be used in combination with a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor or co-stimulatory agonist. In some such embodiments of the invention, the antibody molecule that specifically binds to a checkpoint inhibitor is an anti-PD-1 antibody. Anti-PD1 antibodies are believed to block the inhibitory signal mediated by PD-L1, primarily in CD8 ⁺ T cells; thereby allowing for an enhancement of the T cell-mediated anti-tumor response. These treatments may interact with each other. The same is true for other checkpoint inhibitors and agonist co-stimulatory antibodies.

In addition, the agonist non-blocking TNFR2 antibody molecules described above can be used in combination with other anti-cancer drugs such as chemotherapy (e.g., but not limited to, doxorubicin, paraplatin, cyclophosphamide, paclitaxel, gemcitabine, 5-fluorouracil, docetaxel, vincristine, mitoxantrone, mutamycin, epirubicin, and methotrexate), small molecule tyrosine kinase or serine/threonine kinase inhibitors (e.g., but not limited to, ibrutinib, imatinib, suntinib, regorafenib, sorafenib, dasatinib, erlotinib, vandetanib, midostaurin, vemurafenib, dabrafenib, palbociclib, ribociclib, trametinib, or alectinib), inhibitors targeting growth factor receptors (e.g. but not limited to drugs targeting EGFR/HER1/ErbB1, EGFR2/HER2/ErbB2, EGFR3/HER3/ErbB3, VEGFR, PDGFR HGFR, RET, insulin-like growth factor IGFR, FGFR), antiangiogenic agents (e.g. but not limited to bevacizumab, everolimus, lenalidomide, thalidomide, ziv-aflibercept) or radiation. Generally, all of the above anticancer drugs induce cancer cell death, which results in neoantigen exposure and inflammation. Synergistic effects of the anticancer drug may occur during neoantigen exposure and inflammatory cell influx into the tumor.

A person skilled in the art of medicine may be aware that drugs can be modified by various additives, for example, to change the rate at which the drug is absorbed into the body; and can be modified in various forms, for example, to provide for a particular route of administration into the body.

Accordingly, this application includes the agonist non-blocking antibody molecules, nucleotide sequences, plasmids, viruses and/or cells described herein may be combined with a pharmaceutically acceptable vehicle, carrier, diluent, vehicle and/or adjuvant into a pharmaceutical composition. In this context, the term pharmaceutical composition may be used interchangeably with the terms pharmaceutical preparation, pharmaceutical formulation, therapeutic composition, therapeutic drug, therapeutic formulation and therapeutic entity.

The pharmaceutical compositions described herein may comprise or, in some embodiments of the invention, consist of antibody molecules, nucleotide sequences, plasmids, viruses, or cells.

The pharmaceutical compositions described herein may, in some embodiments of the invention, consist of or contain plasmids comprising nucleotide sequences encoding the antibody molecules described above or comprising the nucleotide sequences described above.

In some embodiments of the invention, the pharmaceutical compositions may comprise nucleotide sequences encoding portions or a complete antibody molecule as described herein integrated into a cellular or viral genome or into a viriom. The pharmaceutical composition may then comprise a cell or virus as a vehicle for delivering an antibody of the invention (or a vehicle for delivering a nucleotide sequence encoding an antibody of the invention). For example, in an embodiment of the invention, the virus may be in the form of a therapeutic oncolytic virus comprising nucleotide sequences encoding at least one of the antibody molecules described herein. In some embodiments, such an oncolytic virus comprises nucleotide sequences encoding a full-length human IgG antibody. In some embodiments, such an oncolytic virus comprises nucleotide sequences encoding an scFv, Fab, or F(ab') ₂ antibody molecule.

As described in the appended claims, some embodiments of the present invention relate to a virus comprising a nucleotide sequence of the present invention or a plasmid of the present invention. Preferably, the virus is an oncolytic virus, such as a therapeutic oncolytic virus. Such oncolytic viruses are known to those skilled in the art of medicine and virology.

In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding an amino acid sequence having at least 80% identity with the sequence set forth in Table 1 above. In some embodiments of the invention, such an oncolytic virus comprises an amino acid sequence having at least 85% identity with the sequence set forth in Table 1 above. In some embodiments of the invention, such an oncolytic virus comprises an amino acid sequence having at least 90% identity with the sequence set forth in Table 1 above. In some embodiments of the invention, such an oncolytic virus comprises an amino acid sequence having at least 95% identity with the sequence set forth in Table 1 above.

In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 7 and ID NO: 8. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 15 and ID NO: 16. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 23 and ID NO: 24. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 31 and ID NO: 32. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 39 and ID NO: 40. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ. ID NO: 47 and ID NO: 48. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 55 and ID NO: 56. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 63 and ID NO: 64. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 71 and ID NO: 72. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 79 and ID NO: 80. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 87 and ID NO: 88. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 95 and ID NO: 96. In some embodiments of the present invention, such an oncolytic virus comprises nucleotide sequences encoding SEQ ID NO: 103 and ID NO: 104.

In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding amino acid sequences having at least 80% identity to the sequence set forth in Table 1 above. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding amino acid sequences having at least 85% identity to the sequence set forth in Table 1 above. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding amino acid sequences having at least 90% identity to the sequence set forth in Table 1 above. In some embodiments of the invention, such an oncolytic virus comprises nucleotide sequences encoding amino acid sequences having at least 95% identity to the sequence set forth in Table 1 above.

As an example, the nucleotide sequence encoding antibody 001-F02 may be as shown in Table 4.

Table 4. Example of nucleotide sequences encoding antibody 001-F02 - the parts of the sequences that are underlined in the table encode the VH and VL sequences, respectively, 001-F02

Coding Subsequence SEQ ID NO: 001-F02 VH
CCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 222 001-F02 VL
CCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGA CCACCACACCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGAACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGA 223

Some oncolytic viruses are able to accept DNA inserts large enough to allow integration of full-length human antibody sequences. Attenuated vaccinia and herpes simplex viruses are examples of therapeutic oncolytic viruses whose genomes are large enough to integrate full-length IgG antibody sequences (Chan, W.M. et al 2014 Annu Rev Virol 1(1):119-141; Bommareddy, P.K., et al. 2018 Nat Rev Immunol 18(8):498-513). Full-length IgG antibodies have been successfully integrated into the oncolytic Vaccinia virus, resulting in the expression and extracellular release of full-length IgG antibodies upon infection of virus-sensitive host cells, such as cancer cells (Kleinpeter, P., et al. 2016, Oncoimmunology 5(10): e1220467). Adenoviruses can also be engineered to encode full-length IgG antibodies that are functionally produced and secreted upon cellular infection (Marino, N., et al. 2017 J Clin Invest 123(6):2447-2463).

The invention also encompasses pharmaceutical compositions comprising a virus, such as an oncolytic virus as discussed above, and a pharmaceutically acceptable diluent, carrier and/or adjuvant.

The pharmaceutical composition in some embodiments of the present invention may be in the form of a CAR-T cell carrying portions or complete antibody sequences described herein as part of the sequence encoding its chimeric T cell receptor antigen.

The invention also encompasses pharmaceutical compositions comprising a CAR-T cell as discussed above and a pharmaceutically acceptable diluent, carrier and/or adjuvant.

The invention also includes other therapeutic methods or "forms" of drugs, such as antibody-drug conjugates, fusion proteins, etc., and a pharmaceutical composition containing such therapeutic methods.

The antibody molecules, nucleotide sequences, plasmids, viruses, cells, and/or pharmaceutical compositions described herein may be suitable for parenteral administration, including aqueous and/or non-aqueous sterile injection solutions that may contain antioxidants and/or buffers and/or bacteriostats and/or solutes that make the composition isotonic with the blood of the intended recipient; and/or aqueous and/or non-aqueous sterile suspensions that may include suspending agents and/or thickening agents. The antibody molecules, nucleotide sequences, plasmids, cells and/or pharmaceutical compositions described herein may be presented in single or multiple dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (i.e., lyophilized) state requiring the addition of only a sterile liquid carrier, such as water for injection, immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders and/or granules and/or tablets of the type previously described.

When administered parenterally to human patients, the daily dose level of the anti-TNFR2 antibody molecule is typically 1 mg/kg of the patient's body weight to 20 mg/kg, and in some cases even up to 100 mg/kg, administered in a single or divided dose. Lower doses may be used in special circumstances, such as in combination with continuous administration. In any case, the physician will determine the actual dosage that will be most suitable for each individual patient, which will vary depending on the age, weight and response of the individual patient. The doses given above are typical for the average case. Of course, there may be individual cases where higher or lower dosage ranges are justified, and such cases are within the scope of the present invention.

Typically, the pharmaceutical composition (or medicament) comprising an antibody molecule described herein will contain the anti-TNFR2 antibody molecule at a concentration of between about 2 mg/mL and 150 mg/mL or between about 2 mg/mL and 200 mg/mL.

Generally, for humans, oral or parenteral administration of the antibody molecule, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein is the preferred and most convenient route. For veterinary use, the antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein are administered in a suitable acceptable formulation in accordance with normal veterinary practice, and a veterinary surgeon will determine the dosage regimen and route of administration that will be most suitable for a particular animal. Accordingly, the present invention relates to a pharmaceutical composition comprising an amount of an antibody molecule, nucleotide sequence, plasmid, virus and/or cell of the present invention effective for the treatment of various conditions (as described above and further below). It is desirable that the antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein are adapted for delivery via a route selected from the group consisting of: intravenous (IV or i.v.); intratumoral (IM or i.m.); intramuscular (SC or i.m.) or subcutaneous.

The present invention also includes antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein, including pharmaceutically acceptable acid addition salts or base addition salts of the target binding molecules or portions thereof of the present invention. Acids which are used to prepare pharmaceutically acceptable acid addition salts of the above-mentioned basic compounds useful in the present invention are acids which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, in particular such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, lactate, citrate, hydrogen citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e. 1,1'-methylene bis-(2-hydroxy-3 naphthoate)] salts. Pharmaceutically acceptable base addition salts can also be used to prepare pharmaceutically acceptable salt forms of the agents of the present invention. Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base salts of the present agents that are acidic in nature are chemical bases that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, non-toxic base salts derived from pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium, or water-soluble amine addition salts such as N-methylglucamine-(meglumine), as well as including lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines. The antibody molecules, nucleotide sequences, plasmids, viruses and/or cells described herein may be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, pellet drying) and/or resuspension methods may be used. Those skilled in the art will appreciate that lyophilization and resuspension may result in varying degrees of loss of antibody activity by the body (e.g., when using conventional immunoglobulins, IgM antibodies tend to lose more activity than IgG antibodies) and that application levels may be adjusted upward to compensate for this. In one embodiment of the present invention, the lyophilized (freeze-dried) polypeptide-binding fragment loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (before lyophilization) upon rehydration.

The anti-TNFR2 antibody molecules, nucleotide sequences, and pharmaceutical compositions described herein can be used to treat cancer in a subject or patient. The terms subject and patient are used synonymously herein.

The term "patient" (or subject) as used herein refers to an animal, including a human, that has been diagnosed with cancer and/or exhibits symptoms of a particular disease.

In some embodiments of the present invention, the patient (or subject) is an animal, including a human, diagnosed with cancer.

In some embodiments of the invention, the patient (or subject) is an animal, including a human, diagnosed with a chronic inflammatory disease and/or exhibiting symptoms of a chronic inflammatory disease. Chronic inflammatory diseases as used herein include autoimmune diseases. As described above, several different immune cells can express TNFR2, and the relative expression levels can vary depending on the disease and context. It is well known that, for example, regulatory T cells can express high levels of TNFR2, and that they can be increased by TNFR2 agonists. Regulatory T cells are a subset of T cells that can suppress other immune cells under normal and pathological immune conditions, and are considered tools for preventing autoimmune attacks on self-tissues. Therefore, stimulation of regulatory T cell activity may be very important in the treatment of autoimmune diseases (Sharabi et al. “Regulatory T cells in the treatment of disease”. Nat Rev Drug Discov. 2018 Oct 12). Examples of chronic inflammatory diseases, in addition to autoimmune diseases, are osteoarthritis and celiac disease. Examples of autoimmune diseases are rheumatoid arthritis (RA), multiple sclerosis (MS), type 1 diabetes mellitus, systemic lupus erythematosus (SLE), psoriasis, inflammatory bowel disease (IBD) or myasthenia gravis (MG).

In some embodiments of the invention, the patient (or subject) is a patient with high expression of TNFR2 in diseased tissue. In this context, high expression means a higher level of expression of TNFR2 compared to the corresponding healthy tissue. Typically, the healthy tissue used for such comparison is a reference tissue (or standard reference) collected from healthy tissue from one or more healthy individuals. The expression level can be measured by standard methods, such as immunohistochemistry (IHC), fluorescence-activated cell sorting (FACS), or mRNA expression measurements.

In this application, it is contemplated that the patient may be a mammal or a non-mammal. Preferably, the mammalian patient is a human, a horse, a cow, a sheep, a pig, a camel, a dog, or a cat. Most preferably, the mammalian patient is a human.

By "demonstrate" in this application is meant that the patient exhibits a symptom of cancer and/or a diagnostic marker of cancer; and/or the symptom of cancer and/or the diagnostic marker of cancer is measurable and/or assessable and/or quantifiable.

A medical professional will immediately understand what the symptoms of cancer and diagnostic markers of cancer are, as well as how to measure and/or assess and/or quantify whether there is a decrease or increase in the severity of cancer symptoms or a decrease or increase in diagnostic markers of cancer; and how these cancer symptoms and/or diagnostic markers of cancer can be used to form a prognosis for the course of cancer.

Cancer treatment is often given as a course of treatment, meaning the drug is given over a period of time. The length of the course of treatment will depend on a number of factors, which may include, but are not limited to, the type of drug being given; the type of cancer being treated; the severity of the cancer being treated; and the age and health of the patient.

By "during treatment" in this application is meant that the patient is currently undergoing treatment and/or receiving a medicinal product and/or receiving a course of a medicinal product.

In some embodiments of the present invention, the cancer to be treated in accordance with the present invention is a solid tumor.

Each of the above types of cancer is well known, and the symptoms and diagnostic markers of cancer are well described, as are the drugs used to treat these types of cancer. Accordingly, the symptoms, diagnostic markers of cancer, and drugs used to treat the above types of cancer are known to medical professionals.

Clinical determinations of the diagnosis, prognosis and progression of a large number of cancers are based on specific classifications known as cancer staging. These cancer staging systems work by comparing a number of different diagnostic markers of cancer and symptoms of cancer to provide a diagnosis and/or prognosis and/or progression of cancer. A specialist in oncology will know how to assess the diagnosis and/or prognosis and/or progression of cancer using a cancer staging system and which diagnostic markers of cancer and symptoms of cancer should be used to do so.

By "determining the stage of cancer" in this application is meant determining the stage of cancer according to the Rai classification, which includes stage 0, stage I, stage II, stage III and stage IV, and/or according to the Binet classification, which includes stage A, stage B and stage C, and/or according to the Ann Arbor classification, which includes stage I, stage II, stage III and stage IV.

It is known that cancer can cause abnormalities in cell morphology. These abnormalities often recur in certain types of cancer, meaning that the study of these changes in morphology (also known as histology) can be used in the diagnosis or prognosis of cancer. Methods for imaging samples to study cell morphology and prepare samples for imaging are well known in the art; for example, light microscopy or confocal microscopy.

By "histological examination" in this application is meant the presence of small mature lymphocytes and/or the presence of small mature lymphocytes with a narrow rim of cytoplasm, the presence of small mature lymphocytes with a dense nucleus devoid of clearly visible nucleoli, and/or the presence of small mature lymphocytes with a narrow rim of cytoplasm and/or with a dense nucleus devoid of clearly visible nucleoli, and/or the presence of atypical cells and/or cleaved cells and/or prolymphocytes.

It is well known that cancer results from mutations in the DNA of a cell, which can cause the cell to escape cell death or to proliferate uncontrollably. Therefore, the study of these mutations (also known as cytogenetic testing) can be a useful tool in assessing the diagnosis and/or prognosis of cancer. An example of this is the deletion of the chromosomal location 13q14.1, which is characteristic of chronic lymphocytic leukemia. Methods for studying mutations in cells are well known in the art; for example, fluorescence in situ hybridization (FISH).

By "cytogenetic testing" in this application is meant the testing of DNA in a cell and, in particular, in a chromosome. Cytogenetic testing may be used to detect changes in DNA that may be associated with the presence of refractory cancer and/or recurrent cancer. Such DNA alterations may include: deletions in the long arm of chromosome 13, and/or deletions in the 13q14.1 locus of chromosome 12, and/or trisomy of chromosome 12, and/or deletions in the long arm of chromosome 12, and/or deletions in the long arm of chromosome 11, and/or deletion of 11q, and/or deletions in the long arm of chromosome 6, and/or deletion of 6q, and/or deletions in the short arm of chromosome 17, and/or deletion in 17p, and/or t(11:14) translocation, and/or (q13:q32) translocation, and/or antigen receptor gene rearrangement, and/or BCL2 rearrangements, and/or BCL6 rearrangements, and/or t(14:18) translocations, and/or t(11:14) translocations, and/or (q13:q32), and/or translocations (3:v), and/or translocations (8:14), and/or translocations (8:v), and/or translocations t(11:14) and (q13:q32).

Cancer patients are known to exhibit certain physical symptoms that are often a result of the burden of cancer on the body. These symptoms often recur in the same cancer and therefore may be indicative of the diagnosis and/or prognosis and/or progression of the disease. The medical professional should understand which physical symptoms are associated with which cancers and how the assessment of these physical systems may correlate with the diagnosis and/or prognosis and/or progression of the disease. "Physical symptoms" in this application include hepatomegaly and/or splenomegaly.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The examples below refer to the following figures:

Figure 1 shows that the antibodies of the present invention bind TNFR2. Figure 1A-D: Human antibodies were shown by ELISA to bind to human TNFR2 protein in a dose-dependent manner, generating different EC50 values. Figure 1E: Murine antibodies 3-F10 and 5-A05 bind to mTNFR2 with similar affinity.

Figure 2 shows the binding of TNFR2-specific n-CoDeR® antibodies to in vitro activated CD4 ⁺ T cells. Human blood-derived CD4 ⁺ T cells (Figure 2A-D) and mouse spleen-derived CD4 ⁺ T cells (Figure 2E) were activated with IL-2 and CD3/CD28 Dynabeads®. The affinity of TNFR2-specific n-CoDeR® antibodies to activated cells was analyzed by FACS at concentrations ranging from 0.002-267 nM (human) and 0.00003-133 nM (mouse). The curves show the MFI after subtraction of isotype control background (Fig. 2 A (complete and partial blockers), Fig. 2 B (partial blockers), Fig. 2 C and D (non-blockers), Fig. 2 E (surrogate mouse complete blocker (3-F10) and non-blocker (5-A05)).

While human TNFR2 antibodies bind with variable affinity to in vitro activated CD4 ⁺ T cells (EC50 values ranging from 0.59 to 53 nM), mouse TNFR2 antibodies bind with similar affinity (EC50 values ranging from 0.072 to 0.11 nM).

Figure 3 shows that TNFR2 n-CoDeR® antibodies specifically bind to TNFR2. Human blood-derived CD4 ⁺ T cells (Figure 3A) and mouse spleen-derived CD4 ⁺ T cells (Figure 3B) were activated for 3 days with recombinant IL-2 and CD3/CD28 activator beads. In vitro activated cells were blocked with anti-TNFR2 polyclonal antibody (gray line) or maintained in PBS (black line) for 30 min before staining with suboptimal concentrations of various TNFR2 n-CoDeR® antibodies or isotype control (dashed line) for 15 min. Cells were then washed and incubated with APC-conjugated secondary antibody for 30 min before flow cytometric analysis.

Binding of all antibodies could be blocked by the TNFR2 polyclonal antibody, therefore, the TNFR2 n-CoDeR® antibodies (human and mouse) were shown to be specific for TNFR2.

Fig. 4 shows the cross-reactivity of human TNFR2-specific n-CoDeR® antibodies to Cynomolgus.

CD4 ⁺ T cells were isolated from cynomolgus monkey blood and stimulated with PMA and ionomycin. After 2 days, cells were labeled with 0.1, 1, or 10 μg/ml TNFR2-specific n-CoDeR® antibodies or isotype control, followed by incubation with APC-conjugated human α secondary antibody. Cells were analyzed by flow cytometry. The figure shows the percentage of TNFR2 ⁺ T cells for individual antibodies compared to isotype control. Results represent the mean and standard deviation (SD) of 2-3 separate experiments.

Most anti-TNFR2 antibodies exhibit cross-reactive binding to cynomolgus cells.

Fig. 5. Activated cells were blocked with 40 μg/ml MR2-1 antibody (Fig. 5 A, black bars) or left in PBS (Fig. 5 A, gray bars) for 30 min, then TNFR2-specific n-CoDeR®/polyclonal TNFR2 (pTNFR2) antibodies were added and the cells were incubated for 15 min. The percentage of bound TNFR2 n-CoDeR® antibodies was analyzed by FACS after incubation with APC-conjugated secondary antibodies. In Fig. 5B activated CD4 ⁺ T cells were blocked with 40 μg/ml TNFR2-specific antibody n-CoDeR®/pTNFR2 (black bars) or maintained in PBS (gray bar) and then incubated for 15 min with PE-conjugated MR2-1 antibody. Cells were then analyzed by FACS.

The MR2-1 antibody did not interfere with the binding of TNFR2 specific n-CoDeR® antibodies, and n-CoDeR® antibodies did not affect the binding of MR2-1 to activated cells, indicating that all n-CoDeR® antibodies bind different domains of the TNFR2 protein than the MR2-1 antibody. This demonstrates that all TNFr2 specific n-CoDeR® antibodies bind different epitopes on the TNFR2 protein than the MR2-1 TNFR2 clone.

Human blood-derived CD4 ⁺ T cells were stimulated with rhIL-2 and CD3/CD28 activation beads for 2-3 days. Activated cells were blocked with 40 μg/ml MR2-1 antibody (Fig. 5A, black bars) or maintained in PBS (Fig. 5A, gray bars) for 30 min, then TNFR2-specific n-CoDeR®/polyclonal TNFR2 (pTNFR2) antibodies were added and the cells were incubated for 15 min. The percentage of bound TNFR2 n-CoDeR® antibodies was analyzed by FACS after incubation with APC-conjugated secondary antibodies. In Fig. 5B activated CD4 ⁺ T cells were blocked with 40 μg/ml TNFR2-specific antibody n-CoDeR®/pTNFR2 (black bars) or maintained in PBS (gray bar) and then incubated for 15 min with PE-conjugated MR2-1 antibody. Cells were then analyzed by FACS.

The MR2-1 antibody did not interfere with the binding of TNFR2 specific n-CoDeR® antibodies, and n-CoDeR® antibodies did not affect MR2-1 binding to activated cells, indicating that all n-CoDeR® antibodies bind different domains of the TNFR2 protein than the MR2-1 antibody.

Figure 6 shows data for ligand blocking antibodies. Blocking ELISA was performed with the hTNFRII-specific n-CoDeR® mAb to assess ligand blocking properties. Figure 6 A) All antibodies were incubated at 10 µg/ml. All antibodies that reduced the signal achieved by the isotype control by more than 50% (indicated by the dotted line) were then dosed to further study the ligand blocking potential. Figure 6 B) shows the complete blocking mAbs, Figures 6 C) and D) show the partially blocking mAbs, and Figure 6 E) shows the weak blocking mAbs. All other mAbs are considered non-blocking mAbs.

Figure 7 shows data for ligand blocking antibodies. Blocking ELISA was performed with the mTNFRII-specific n-CoDeR® mAb to assess ligand blocking properties. Figure 7 A) All antibodies were incubated at 10 μg/mL. All antibodies that reduced the signal achieved by the isotype control by more than 50% (indicated by the dotted line) were then dosed to further study the ligand blocking potential. Figure 7 B) shows the complete blocking mAbs, Figures 7 C) and D) show the partially blocking mAbs, and Figure 7 E) shows the weak blocking mAbs. All other mAbs are considered non-blocking mAbs.

Figure 8 shows that agonist non-blocking but not antagonist TNFR2 blocking n-CoDeR® specific antibodies enhance IFN-γ production in NK cells stimulated with IL-2 and IL-12. Figure 8A: Human blood-derived NK cells were stimulated with 20 ng/ml rhIL-2 and 20 ng/ml rhIL-12 supplemented with 10 μg/ml TNFR2-specific n-CoDeR® antibodies, isotype control, or 100 ng/ml rhTNF-α for 24 h. The amount of IFN-γ in culture supernatants was measured by MSD. The amount of IFN-γ was normalized to the isotype control and shown in Figure 8A. 8 A. Human antibodies that had an EC50 value higher than 25 nM against in vitro activated CD4 ⁺ T cells were not included in the analysis. Human NK cells also produce TNF-α in these cultures (see Fig. 8D below). The IFN-γ results represent the average of 3 donors in 2 independent experiments. The results show that non-blocking TNFR2 antibodies are agonists and increase IFN-γ production from cytokine-stimulated NK cells, whereas blocking antibodies are rather antagonistic and decrease IFN-γ production by NK cells. Furthermore, Fig. 8 BD shows that agonist antibodies have intrinsic agonist activity even in the absence of measurable TNF-α ligand. As shown in Fig. 8B, the addition of TNF-α blocking antibody reduces the amount of IFN-γ released, but the ratio compared to the isotype control (Fig. 8C) is still maintained even in the complete absence of measurable TNF-α in the supernatant (Fig. 8D). Fig. 8D shows the mean TNF-α levels in 2 donors in the presence or absence of TNF-α neutralizing antibody.

Figure 9 shows that agonistic non-blocking, but not antagonistic TNFR2-specific blocking n-CoDeR® antibodies activate the memory CD4 ⁺ T cell population, as evidenced by an increase in the proportion of CD25 ⁺ cells. Human blood-derived CD4 ⁺ T cells (Figure 9 A) and mouse spleen CD4 ⁺ T cells (Figure 9 B) were activated with recombinant IL-2 and TNFR2-specific n-CoDeR® antibodies, isotype control, or recombinant TNF-α. After 3 days of culture, cells were stained for CD25 and CD45RO (human)/CD44 and CD62L (mouse) and analyzed by flow cytometry. Results show the percentage of CD25-expressing cells in the memory population (CD45RO ⁺ (human)/CD44 ^{+ CD62L−} (mouse)) compared with the percentage of CD25 ⁺ cells isolated in isotype control cultures. Results represent the mean and SEM for 7 donors (Fig. 9A, human) and 3 mice (in 2 independent experiments) (Fig. 9B). In both human and mouse cultures, non-blocking TNFR2 antibodies induced a percentage of CD25 ⁺ memory cells, whereas blocking antibodies had no effect on the memory population. For both human (Fig. 9A) and mouse (Fig. 9B) cultures, addition of exogenous TNF-α increased the CD25 ⁺ memory T cell population. * = p < 0.05 as calculated by one-way ANOVA.

Figure 10 shows that anti-TNFR2 mAbs modulate the suppressive function of myeloid-derived suppressive cells (MDSC). For Figures 10 A and B, MDSC were pre-incubated with human anti-TNFR2 mAb for 30 min. Then, without washing the cells, titratable amounts of MDSC were incubated with CFSE-labeled CD3 ⁺ T cells in the presence of CD3/CD28 Dynabeads®. Figure 10A: % of activated CD25 ⁺ T cells were determined after 3 days by FACS. To normalize between different assays, isotype control background was subtracted from all data points. Figure 10 shows the sum of 6 different donors in 3 independent experiments at a 1:4 MDSC to T cell ratio. 10B: The amount of secreted cytokines in the supernatant was estimated by MSD and the ratio of IFN-γ and IL-10 release relative to the isotype control was calculated. Data were pooled from analysis of culture supernatants obtained from experiments with 5 different donors.

For Fig. 10, CD11b ⁺ myeloid cells were isolated from a CT26 murine tumor and pre-incubated with murine anti-TNFR2 mAb for 30 min. Titrating numbers of myeloid cells were co-cultured with CFSE-labeled CD3+ T cells that were purified from naïve Balb/C spleens. Cells were stimulated with CD3/CD28 Dynabeads® for 3 days, and then the % of proliferating CFSE ^low cells was analyzed by FACS (n = 4 independent experiments). Only agonist/non-blocking anti-TNFR2 mAbs were shown to abolish MDSC suppressive function in T cell/MDSC co-culture assays.

In Fig. 10 D, MDSC were pre-incubated for 30 min with two different human anti-TNFR2 mAbs (one blocking = 1H10) and one non-blocking (= 1F02) in 3 different isotypes, including a defective Fc format, amino acid position 297 was switched, resulting in loss of glycosylation and thus reduced binding to FcγR (herein designated as N297Q). Without washing the cells, titratable amounts of MDSC were then incubated with CFSE-labeled CD3 ⁺ T cells in the presence of CD3/CD28 Dynabeads®, and the % of activated CD25 ⁺ T cells was determined after 3 days by FACS. To normalize between different assays, the isotype control background was subtracted from all data points. The figure shows that the agonist activity of the non-blocking antibody 1-F02 is independent of antibody isotype and FcγR binding. The figure shows the sum of 4 different donors in 2 independent experiments at a 1:4 MDSC to T cell ratio.

Fig. 11. For Fig. 11, Balb/c mice were injected subcutaneously with 1 x 10 ⁶ CT26 cells. After 8 days, when the average tumor size was 3 x 3 mm, the mice were treated twice weekly with 10 mg/kg antibody intraperitoneally as shown in the figures. Tumors were measured twice weekly until they reached 15 mm in diameter, at which time the mice were sacrificed. The top figure shows tumor growth in mice treated with an isotype control, then the two figures below, on the left panel, show a ligand-blocking antagonist antibody (middle figure) and a non-ligand-blocking agonist antibody (lower figure), in the FcγR-deficient Ig format. The middle panel shows the same antibodies in the mouse IgG2a format, which primarily engages activating FcγR, and the right panel shows the antibodies in the mouse IgG1 format, which primarily engages inhibitory FcγRIIb. In Fig. 11B, surviving mice were followed for 70 days. As can be seen in the figures, the non-blocking agonist antibody is most effective in treating tumors in the IgG1 format, which primarily engages inhibitory FcγR. In addition, the agonist antibody has an FcγR-independent antitumor effect, as seen with the N297A format. On the other hand, the blocking antagonist antibody is most effective in treating tumors in the IgG2a format, which primarily engages activating FcγR, and has no effect in the FcγR binding-deficient format. *** = p < 0.001 compared to isotype control calculated using the Mantel-Cox log-rank test.

Fig. 12. C57/BL6 mice were injected subcutaneously with 1 × 10 ⁶ MC38 cells. At an average tumor size of 3 × 3 mm, mice were treated twice weekly with 10 mg/kg antibody intraperitoneally as shown in the figures. The figure shows tumor growth curves in individual mice. Fig. 12 A: isotype control, Fig. 12 B: PD-1 targeted antibody, Fig. 12 C: 5A05 antibody (surrogate antibody, non-blocker ligand, agonist), Fig. 12 D: combination of 5A05 and PD1. Tumors were measured twice weekly until they reached a diameter of 15 mm, at which time mice were sacrificed. In Fig. 12E shows survival curves for four different treatment groups, * = p < 0.05, *** = p < 0.001 compared with isotype control, calculated using the Mantel-Cox log-rank test.

Figure 13 shows that ligand blocking agonist antibodies are effective as antitumor treatment in combination with anti-PD-L1. C57/BL6 mice were injected subcutaneously with 1 x 10 ⁶ MC38 cells. At an average tumor size of 5 x 5 mm, mice were treated twice with an isotype control antibody or 5A05 (days 1 and 4), or four consecutive days with anti-PD-L1 followed by a fifth injection two days later (for a total of five injections on days 1, 2, 3, 4, and 7), or a combination of both. All antibodies were administered at a dose of 10 mg/kg intraperitoneally. Figure 13 shows mean tumor growth +/- SEM, n = 10/group. * = p < 0.05, *** = p < 0.001, as calculated using one-way ANOVA.

Fig. 14. C57/BL6 mice were injected subcutaneously with 1 × 10 ⁶ B16 cells. Three days later, mice were injected intraperitoneally with 10 mg/kg antibody twice weekly. Tumors were measured twice weekly until they reached 15 mm in diameter, after which mice were sacrificed. Fig. 14A: isotype control, Fig. 14B: 5A05 antibody (surrogate antibody, non-ligand blocker, agonist). Fig. 14C shows the survival curves for the two different treatment groups. * = p < 0.05 compared with isotype control calculated by the Mantel-Cox log-rank test.

Fig. 15. Non-blocking ligand agonist surrogate antibody 5A05 alters immune cell composition in tumors. Mice were inoculated with CT26 tumor cells as described and treated with antibodies as indicated after tumors reached approximately 7 x 7 mm in size after 3 injections. At day 8 after treatment initiation, mice were sacrificed and tumors were harvested. Single cell tumor suspensions were analyzed for immune cell content by FACS. A) Non-blocking ligand agonist surrogate antibody 5A05 induces Treg depletion and B) CD8 ⁺ T cell influx or expansion. This results in a shift in the Treg/CD8 ⁺ T cell ratio as shown in C) Fig. D) shows that not only T cells but also myeloid cells, here the number of tumor-associated macrophages (TAM, defined as CD11b ⁺ but negative for both Ly6G and Ly6C) is very significantly reduced. The ligand blocking agonist surrogate antibody 3F10 also modulates the number of TAMs, but still significantly different from 5A05.

Fig. 16. NOG mice were injected i.v. with 15-20 × 10 ⁶ PBMCs. After 10-12 days, the mice were spleen-removed, single-cell suspensions were prepared, and TNFR2 expression was assessed by FACS. TNFR2 expression was previously assessed on T cells obtained from blood and tumor samples from 3 or 9 cancer patients, respectively. As shown in the figure, TNFR2 expression on Tregs and CD8 ⁺ T cells is highly comparable between human T cells expanded and activated in vivo in NOG mice and T cells from human tumors.

Fig. 17. NOG mice were injected i.v. with 15-20 × 10 ⁶ PBMCs. After 10-12 days, the mice's spleens were removed, single cell suspensions were prepared, and the cells were analyzed by FACS. Fig. 17A shows the mean percentage of stained Tregs, defined as CD45 ⁺ CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127low ^/neg , out of total cells in the spleen. Fig. 17B shows the mean percentage of T cells (CD3 ⁺ ) out of total cells in the spleen. As shown in Fig. 17, the non-ligand blocking agonist antibody 1F02 increased the number of Tregs (expressing the highest levels of TNFR2) as well as the total number of T cells in this model. In comparison, the ligand blocking antagonist antibody 1H10 had no such effect.

Fig. 18. IFN-γ release induced by different TNFR2-specific antibodies was measured in three different in vitro systems. The anti-CD3 antibody OKT3 or Muromonab-CD3, the anti-CD52 antibody alemtuzumab, and the anti-CD28 antibody were used as positive controls. The isotype control was used as a negative control. Each point represents PBMCs from a single human donor. Fig. 18A shows the results for high-density cell cultures where PBMCs were cultured at a concentration of 1 × 10 ⁷ cells/mL. After 48 h, 10 μg/mL antibody was added and incubated for 24 h. As can be seen in the figure, both alemtuzumab and OKT3 induced significant IFN-γ release but did not induce any TNFR2-specific antibodies. Fig. 18B shows in vitro solid phase cultures prepared by coating the wells of a 96-well plate with antibodies prior to addition of PBMCs. Again, both alemtuzumab and OKT3 induced significant release of IFN-γ along with some TNFR2 specific antibodies, particularly in one of the donors. Fig. 18C shows whole blood stimulation with antibody, and here alemtuzumab induced significant release of IFN-γ but not any TNFR2 specific antibodies.

Fig. 19: NOG mice were injected i.v. with 25 x 10 ⁶ PBMC cells. After 14 days, when the blood of mice was shown to consist of approximately 40% human T cells, mice were treated with 10 μg of antibody. Body temperature was measured 1 hour after injection (Fig. 19 A). The experiment was terminated 5 hours after injection, and blood was analyzed for IFN-γ (Fig. 19 B) or TNF-α (Fig. 19 C). **** = p < 0.0001 and ** = p < 0.01, as calculated by one-way ANOVA.

Figure 20 shows binding to TNFR2 variants lacking individual domains. Antibody binding to TNFR2 variants expressed on HEK cells was tested by flow cytometry. The absence of domains 1 and 2 does not significantly affect binding (Figure 20 A and B), whereas 3 and partially 4 completely abolish the interaction between the antibody and TNFR2 (Figure 20 C and D). Similarly, the absence of domain 1 + 3 completely prevents binding of all antibodies (except 1F06) (Figure 20 E), whereas the absence of domain 2 + 4 completely abolishes binding of agonist antibodies (1F02, 1F06, 4E08) and significantly reduces binding also to antagonists (1H10, 4H02, 5B08) (Figure 20 F). Dark gray color indicates positive control and white color indicates negative control antibody.

Figure 21 shows a comparison of the amino acid sequence of domain 3 of human (H-D3) and mouse (M-D3) TNFR2. Similar amino acids are highlighted in white and differences are highlighted in gray. The five sequences below represent the five different constructs against which antibodies are tested. Sequence exchanges from human to mouse are underlined, while the unmarked sequence is fully human. Domains 1, 2, and 4 are human and contain no substitutions or mutations.

Fig. 22. Binding to wild-type human and mouse TNFR2 is shown in the left panel. Mutated hTNFR2 constructs (m1, m2, m3, and m4) were used to narrow the binding site for different anti-hTNFR2 antibodies. Flow cytometry analysis showed that mutations at amino acids 119–132 did not affect antibody binding, but at amino acids 151–160 completely abolished binding of all antibodies. Mutations at 134–144 impaired binding of only blocking and antagonist antibodies but had no significant effect on agonist antibodies. Dark gray bars indicate positive controls and white bars indicate negative controls. The dotted line indicates the level of negative control antibody.

EXAMPLES

Specific examples, not intended to be limiting, will now be described which incorporate certain aspects of the invention.

In many examples, particularly in vivo examples, the antibody 5-A05 was used. This is a murine antibody that is a surrogate antibody for the human antibodies described herein. It was chosen as a surrogate antibody based on its characteristics similar to those of a non-ligand blocking agonist antibody with a good EC50 value.

In some examples and figures, slightly different names are used for antibody clones, for example, clone 001-F02 is sometimes shortened to 1-F02 or 1F02, 005-B08 is sometimes shortened to 5-B08 or 5B08, etc.

Example 1 - Production of antibodies specific to TNFR2

(See also Fig. 1 and the above description of this figure.)

Isolation of scFv antibody fragments

The n- ^Coder® scFv library (BioInvent; Söderlind E, et al. Nat Biotechnol. 2000;18(8):852-6) was used to isolate scFv antibody fragments recognizing human or mouse TNFR2.

The phage library was used in three consecutive pannings against recombinant human or mouse protein (Sino Biological). After phage incubation, cells were washed to remove unbound phage. Binding phage were eluted with trypsin and amplified in E. coli. The resulting phage stock was converted to scFv format. E. coli was transformed with plasmids carrying scFv, and individual scFv clones were expressed.

Identification of a unique TNFR2 binding scFv

Converted scFvs from the third panning were analyzed using the FMAT homogeneity assay (Applied Biosystems, Carlsbad, CA, USA) for binding to 293 FT cells transfected to express human or mouse TNFR2 or an unrelated protein.

Briefly, transfected cells were added to clear-bottomed plates along with scFv-containing supernatant from expression plates (diluted 1:7), mouse anti-His Tag antibody (0.4 μg/ml; R&D Systems), and APC-conjugated goat anti-mouse antibody (0.2 μg/ml; cat. no. 115-136-146, Jackson Immunoresearch). FMAT plates were incubated at room temperature for 9 h before reading. Bacterial clones binding to TNFR2-transfected cells but not to cells transfected with an unrelated protein were classified as active and selectively collected into a 96-well plate.

IgG binding to TNFR2 in ELISA

96-well plates (Lumitra 600 LIA plate, Greiner) were coated overnight at 4°C with recombinant human or mouse TNFR2-Fc protein (Sino Biological) at a concentration of 1 pmol/well. After washing, titrated doses of anti-TNFR2 mAb from 20 μg/ml to 0.1 ng/ml (133 nM to 1 pM) were allowed to bind for 1 h. The plates were then washed again and bound antibodies were detected with a secondary anti-human-F(ab)-HRP antibody (Jackson ImmunoResearch) diluted at 50 ng/ml. Super Signal ELISA Pico (Thermo Scientific) was used as a substrate and the plates were analyzed using a Tecan Ultra Microplate reader.

The data shown in Table 5 and Fig. 1A-D indicate that all human anti-TNFR2 antibodies bind to human TNFR2 protein. EC50 values range from 0.082 nM for 1-C08 to 4.4 nM for 1-A09.

In addition, surrogate mouse antibody clones 3-F10 and 5-A05 also bind to the mTNFR2 protein. These two clones bind with very similar affinity (Table 5 and Fig. 1E).

Table 5. EC50 values of antibody binding to TNFR2 protein (human protein, except clone 3F10 and 5A05)

Clone EC ₅₀ (nM) 1-C08 0.082 1-E06 0.20 1-G10 0.29 1-H10 0.29 4-H02 0.20 5-B02 0.15 5-B08 0.17 1-G04 1.7 1-H09 0.30 1-D01 0.37 5-F10 0.22 1-B11 0.25 1-C07 0.26 1-B05 0.23 1-F02 0.31 1-F06 0.15 4-E08 0.38 1-G05 0.54 1-A09 4.4 1-B09 0.18 1-C03 0.75 1-C05 0.38 3-F10 (mouse) 0.97 5-A05 (mouse) 1.4

Example 2 - Antibody specificity

(See also Figs. 2-5 and the above description of these figures.)

CD4 ⁺ T cell isolation

PBMCs from human buffy coat and cynomolgus macaque (M. fascicularis) whole blood were isolated using Ficoll-Paque PLUS gradients (GE Healthcare). CD4 ⁺ T cells were isolated from PBMCs by magnetic cell sorting using the Human CD4 ⁺ T Cell Isolation Kit or non-human primate (cynomolgus macaque) CD4 MicroBeads, both from Miltenyi. Mouse CD4 ⁺ T cells were isolated from the spleen using the Mouse CD4 ⁺ T Cell Isolation Kit from Miltenyi.

Titration of TNFR2-specific antibodies n-CoDeR®

The ability and affinity of TNFR2 n-CoDeR® antibodies to bind TNFR2 expressed on cells were determined using in vitro activated CD4 ⁺ T cells. Human CD4 ⁺ T cells were stimulated with rhIL-2 (50 ng/ml) (R&D systems) and Dynabeads® T Activator CD3/CD28 for T Cell Expansion and Activation (Gibco) for 2-3 days at 37°C. In vitro activated cells were labeled with increasing amounts of TNFR2-specific or isotype control n-CoDeR® antibodies in the range of 0.002-267 nM. Cells were then incubated with APC-conjugated a-human IgG secondary antibody (Jackson) followed by flow cytometry analysis (FACSVerse, BD). The resulting titration curves are shown in Fig. 3A-D. Mouse CD4 ⁺ T cells were stimulated with 135 U/ml rmIL-2 (R&D systems) and Dynabeads® CD3/CD28 T activator for T cell expansion and activation (Gibco) for 2-3 days at 37°C. In vitro activated cells were labeled with increasing amounts of n-CoDeR® antibodies specific for TNFR2 or isotype control ranging from 0.00003-133 nM. Cells were then incubated with a-mouse IgG secondary antibody conjugated to APC (Jackson) followed by flow cytometry analysis (FACSVerse, BD). Titration curves are shown in Fig. 3E. The EC50 values for the titration curves were calculated in Microsoft Excel and are shown in Table 6. For human antibodies, the EC50 values ranged from 0.6 nM (4-H02) to 52.7 nM (1-CO3). Mouse antibodies bound to in vitro activated cells with similar affinity (0.072 nM (3-F10) and 0.11 nM (5-A05)).

Specificity of TNFR2 n-CoDeR® antibodies

The specificity of TNFR2 antibodies to TNFR2 was determined in FACS blocking experiments with commercial polyclonal TNFR2 antibodies (R&D systems). CD4 ⁺ T cells (mouse and human) stimulated for 2-3 days with 50 ng/ml rhIL-2 (R&D systems) (human)/135 U/ml rmIL-2 (R&D systems) (mouse) and Dynabeads® CD3/CD28 T Activator for T Cell Expansion and Activation (Gibco) were blocked with 40 μg/ml TNFR2 polyclonal antibody (R&D systems) for 30 min followed by 15 min incubation with TNFR2 n-CoDeR® antibodies or isotype control. The concentration of n-CoDeR® antibodies used was based on titration curves for individual n-CoDeR® TNFR2 antibodies and a suboptimal concentration was selected for each antibody. Cells were then washed and incubated for 30 min with APC-conjugated secondary antibody (Jackson). Cells were analyzed by flow cytometry (FACSVerse, BD). All binding of TNFR2-specific n-CoDeR® antibodies (both human and mouse) could be blocked by the anti-TNFR2 polyclonal antibody as shown in Fig. 4. These results confirm that TNFR2 n-CoDeR® antibodies specifically bind TNFR2 on CD4 ⁺ T cells activated in vitro.

Epitope mapping of TNFR2-specific n-CoDeR® antibodies against TNFR2 antibody clone MR2-1

The TNFR2 antibody clone MR2-1 (Invitrogen) binds a specific domain of the TNFR2 protein. If the TNFR2-specific n-CoDeR® antibodies bind to the same domain as MR2-1, they were tested by FACS blocking experiments.

Human CD4 ⁺ T cells were stimulated for 2-3 days with 50 ng/ml rhIL-2 (R&D systems) and Dynabeads® CD3/CD28 T cell activator for expansion and activation (Gibco). Activated cells were blocked with 40 μg/ml MR2-1 (black bars in Fig. 5A), TNFR2-specific antibody n-CoDeR®/polyclonal TNFR2 (R&D systems) (black bars in Fig. 6B), or PBS (gray bars in Fig. 5). After 30 min of incubation, cells were immediately stained with TNFR2-specific antibodies n-CoDeR®/pTNFR2 (Fig. 5A) or MR2-1 (Fig. 5B) for 15 min. The cells in Fig. 5A were also incubated with a secondary reagent of human IgG conjugated to APC (Jackson). All cells were analyzed by flow cytometry (FACSVerse, BD). Since the percentage of MR2-1 ⁺ cells was the same for both n-CoDeR® blocked and unblocked cells (Fig. 5B) and the binding of n-CoDeR® antibodies was the same with or without MR2-1 blocker (Fig. 5A), these n-CoDeR® antibodies likely bind different epitopes of the TNFR2 protein than the MR2-1 antibody.

Binding of TNFR2 n-CoDeR® antibodies to cynomolgus monkey

To confirm the cross-reactivity of TNFR2 antibodies to cynomolgus macaque, cynomolgus macaque CD4 ⁺ T cells were stimulated with 50 ng/ml PMA (Sigma) and 100 ng/ml ionomycin (Sigma) for 2 days. The cells were incubated with TNFR2-specific n-CoDeR® antibodies at 3 different concentrations (0.1, 1, and 10 μg/ml) and then incubated with APC-conjugated secondary a-human IgG reagent (Jackson). The cells were analyzed by flow cytometry (FACSVerse, BD), and the result showed that most of the n-CoDeR® antibodies specific for human TNFR2 could bind cynomolgus macaque TNFR2, the results for individual antibodies are shown in Fig. 4.

Thus, the data in Example 2 demonstrate that human antibodies bind specifically to TNFR2 endogenously expressed on human immune cells. Furthermore, the data demonstrate that this binding can be blocked by the addition of a commercially available polyclonal anti-TNFR2 antibody, indicating a very high specificity for TNFR2. The same is true for the surrogate clones 3F10 and 5A05 against mouse cells expressing mouse TNFR2. Furthermore, binding of the human clones is not dependent on MR2-1 antibodies, which exhibit a different epitope specificity than MR2-1. T

Table 6. EC ₅₀ values calculated from titration of TNFR2-specific antibodies to in vitro activated CD4 ⁺ T cells

Clone EC ₅₀ (nM) 1-C08 2.6 1-E06 4.1 1-G10 3.3 1-H10 1,1 4-H02 0.59 5-B02 0.80 5-B08 1,2 1-G04 18 1-H09 16 1-D01 3.9 5-F10 32 1-B11 27 1-C07 36 1-B05 1.5 1-F02 0.79 1-F06 2.5 4-E08 2,3 1-G05 0.66 1-A09 48 1-B09 29 1-C03 53 1-C05 12 3-F10 (mouse) 0.072 5-A05 (mouse) 0.11

Example 3 - Verification of ligand blocking properties

(See also Figs. 6-7 and the above description of these figures.)

IFA method

96-well plates were coated with hTNFRII (Sinobiology cat. nr10414-H08H) or mTNFRII (Sinobiology cat. no. 50128 M08H) at a concentration of 2.5 pmol/well in ELISA coating buffer (0.1 M sodium carbonate, pH 9.5) and incubated overnight at 4°C. After washing in ELISA wash buffer (PBS with 0.05% Tween20), the plates were incubated with slow shaking for 1 h at room temperature with n-CoDeR® monoclonal antibodies at a concentration of 10 μg/ml (single-dose ELISA) or 33 nM, followed by 1:2 dilutions (ELISA titration) in blocking buffer containing 0.45% fish gelatin. Subsequently, recombinant hTNF-α-bio (R&D, Cat. No. BT210) or mTNF-α (Gibco Cat. No. PMC3014) was added at a final concentration of 5 nM and 2 nM, respectively, and incubated for an additional 15 min. The plates were then washed. For human ELISA, streptavidin-HRP (Jackson Cat. No. 016-030-084) diluted 1:2000 in blocking buffer was added and incubated again for 1 h at room temperature, followed by washes first in ELISA buffer and then in Tris buffer (pH 9.8). The substrate (Super Signal ELISA Pico from Thermo Scientific cat. no. 37069) was then diluted according to the manufacturer's instructions, added to the wells and incubated in the dark for 10 min before reading on a Tecan Ultra. For the mouse ELISA, rabbit anti-mTNF-α (Sinobiological cat. no. 50349-RP02) was diluted to 1 μg/ml and allowed to incubate for 1 h at room temperature. After washing, anti-rabbit-HRP diluted 1:10,000 in blocking buffer was added and incubated again for 1 h at room temperature. Substrate addition and reading were performed as above.

Data for anti-human antibodies and anti-mouse antibodies are presented in Tables 7 and 8, respectively, below and in Figs. 6 and 7.

Table 7. EC50 values of human ligand blocking antibodies: antibodies were titrated and EC50 values were calculated

Clone EC50 (nM) Blocking 001-H10 0.9 Complete 004-H02 0.4 Complete 005-B08 0.3 Complete 005-B02 0.2 Complete 001-E06 0.3 Partial 001-G10 1.6 Partial 001-C08 1,1 Partial 001-H09 1.4 Partial 005-F10 0.03 Partial 001-G04 3.2 Partial 001-B11 1.0 Weak 001-C07 0.8 Weak 001-D01 1.4 Weak

Table 8. EC50 values of mouse ligand blocking antibodies: antibodies were titrated and EC50 values were calculated

Clone EC50 (nM) Blocking 3-F10 1.9 Complete 4-C01 2.7 Complete 4-A06 2.0 Partial 4-A07 >500 Partial 4-F06 6.2 Partial 5-C09 8.6 Partial 2-D09 4.4 Partial 4-B12 >500 Partial 3-G06 13 Partial 2-H01 25 Weak 4-C02 >500 Weak 4-G09 2.6 Weak 4-C03 8.3 Weak

Definitions of blockers

- Complete blockers are defined as reducing TNF-α binding by more than 98%

- Partial blockers are defined as reducing TNF-α binding by 60-98%

- Weak blockers are defined as those that reduce TNF-α binding by less than 60%

- Non-blocking antibodies are defined as those not achieving more than 50% blocking in the high-dose single-point ELISA as shown in Figs. 6A and 7A.

The data presented in this example show that a variety of antibodies have been generated, ranging from antibodies that completely inhibit TNF-α ligand from binding to antibodies that do not inhibit ligand blocking at all. This is true for both human antibodies and mouse surrogates.

Example 4 - In vitro functionality of antibodies

(See also Figs. 8-10 and the above description of these figures.)

Non-TNF-α blocking antibodies to TNFR2 enhance cytokine-stimulated IFN-γ production by NK cells, unlike TNF-α blocking TNFR2 antibodies

The agonist/antagonist properties of TNFR2-specific antibodies were assessed using the NK cell assay described by Almishri et al. (TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2. Almishri W.et al. J Innate Immun. 2016;8:617-629).

Briefly, human NK cells were isolated from human PBMCs by MACS using the “NK Isolation Kit” (Miltenyi). 100 μl of NK cells (1 x ¹⁰⁶ cells/ml) were cultured with 20 ng/ml rhIL-2 (R&D systems) and 20 ng/ml rhIL-12 (R&D systems) together with 10 μg/ml TNFR2-specific antibodies, 10 μg/ml isotype control or 100 ng/ml TNF-α (R&D systems) in U-bottom plates (96-well Corning® TC-treated microplates, Sigma-Aldrich). Supernatants were collected after 24 h and the amount of IFN-γ produced was estimated by MSD. As a control, anti-TNF-α antibodies neutralizing TNF-α (Cat. No. AF-210-NA, R&D systems) were used. As shown in Fig. 8D, the 1 μg/ml dose completely neutralized soluble TNF-α and this dose also reduced the release of IFN-γ.

Non-blocking human TNFR2 antibodies markedly enhanced IFN-γ production by IL-2 and IL-12 stimulated NK cells (2-3 times more IFN-γ than isotype control), whereas antagonist antibodies (shown here with full blockers) exhibited antagonistic effects on IFN-γ production by NK cells (Fig. 8A).

This assay was not considered representative for use with mouse surrogate antibodies due to the lack of endogenously produced TNF-α in mouse cultures and the expression of inhibitory FcγR on mouse NK cells and only activation of FcγR on the human counterpart. Instead, a memory T cell activation assay (CD25 induction) and a myeloid-derived suppressor cell (MDSC) suppression assay (both described below) were used to study the agonist or antagonist properties of mouse surrogate antibodies.

Non-ligand blocking but not TNFR2 blocking antibodies induce CD25 expression on memory CD4 ⁺ T cells

To further understand the agonist/antagonist properties of TNFR2 antibodies, their ability to increase the proportion of CD25 expressing memory CD4 ⁺ T cells was assessed.

Briefly, human CD4 ⁺ T cells were isolated from PBMCs by MACS using the “CD4 ⁺ T cell isolation kit” from Miltenyi. CD4 ⁺ T cells were cultured with 10 ng/ml rhIL-2 (R&D systems) and 10 μg/ml TNFR2-specific antibodies or the indicated amount of rhTNF-α (R&D systems). After 3 days, CD25 expression on memory cells (CD45RO ⁺ cells) was analyzed by FACS (Fig. 9A).

Similarly, mouse CD4 ⁺ T cells were isolated from the spleen by MACS using the “CD4 ⁺ T cell isolation kit” (Miltenyi) and cultured with 10 ng/ml rmIL-2 (R&D systems) and 10 μg/ml TNFR2-specific antibody or the indicated amount of rmTNF-α (R&D systems). CD25 expression on memory cells (CD44 ⁺ CD62L ⁻ cells) was analyzed by FACS after 3 days (Fig. 9B).

The percentage of cells expressing CD25 was increased in memory cell cultures stimulated with non-blocking TNFR2, again demonstrating agonist activity in both humans and mice. However, stimulation with blocking antibodies did not increase CD25 expression in these cultures.

A non-ligand blocking but not anti-TNFR2 mAb suppresses myeloid-derived suppressor cell (MDSC) function in T cell/MDSC co-culture assays

The effect of human anti-TNFR2 mAb on MDSC suppressive function was assessed in T cell/MDSC co-culture assays. Briefly, MDSC were generated by culturing MACS-isolated human CD14 ⁺ monocytes in 50% ascites fluid isolated from cancer patients and 50% R-10 for 3 days. MDSC were then washed and pre-incubated with 10 μg/ml anti-TNFR2 mAb for 30 min. Without washing the cells, titratable amounts of MDSC were co-cultured with MACS-isolated, CFSE-labeled CD3 ⁺ T cells in the presence of CD3/CD28 Dynabeads®. After 72 h, the percentage of activated CD25 ⁺ T cells was assessed by FACS. IFN-γ and IL-10 secretion were measured by MSD according to the manufacturer’s instructions.

In contrast to blocking anti-TNFR2 mAbs (dark gray bars), non-blocking anti-TNFR2 mAbs (light gray bars) were shown to increase the percentage of activated CD25 ⁺ in co-culture (Fig. 10A). By measuring the amount of secreted interferon gamma (IFN-γ) and interleukin-10 in the supernatant, the ratio of the two cytokines was used to assess the Th1/Th2 balance. As shown in Fig. 10B, the IFN-γ to IL-10 ratio was significantly increased by non-blocking anti-TNFR2 antibodies compared to blocking antagonist antibodies, indicating that non-blocking antibodies induce a shift toward the Th1 pathway.

Regarding human antibodies, the effect of mouse anti-TNFR2 antibodies was tested in a similar suppression assay. Here, CD11b ⁺ myeloid cells were directly MACS-isolated from CT26 mouse tumors and preincubated for 30 min with anti-TNFR2 mAb. CD3 ⁺ responder T cells were purified from naïve Balb/C spleens. Myeloid suppressor cells and CFSE-labeled T cells were co-cultured at various ratios for 3 days. The percentage of proliferating CFSE ^low T cells was determined by FACS. Similar to the results of human assays, the percentage of proliferating responder cells was significantly lower after incubation with blocking than with non-blocking antibodies (Fig. 10C).

In MDCS suppressor cultures, myeloid cells express high levels of various FcγRs. To test whether the observed agonist and antagonist effects are Fc dependent, the non-ligand blocking agonist antibody 1F02 was tested in several formats, hIgG1, good binding to activating FcγRs, hIgG2, good binding also to the inhibitory defective FcγRIIB format, and Fc with greatly reduced binding to all FcγRs. The results in Fig. 10D show that the agonist activity of the non-blocking antibody 1-F02 is independent of antibody isotype and FcγR binding.

In summary, the data in Example 4 demonstrate that non-ligand blocking antibodies are agonistic as measured by multiple in vitro assays: NK cell-mediated IFN-γ release, CD4 ⁺ memory cell activation measured by CD25 expression and T cell proliferation, and CD25 expression in an MDCS co-culture assay. Furthermore, the data demonstrate that this is an intrinsic characteristic independent of the presence of ligand as well as antibody isotype. We also show that the surrogate murine antibody 5A05 has similar agonistic characteristics. Antibodies that stimulate T cells or NK cells may be useful in cancer treatment and may induce an endogenous immune response, ultimately destroying malignant cells.

In accordance with the invention, non-ligand blocking agonist antibodies are shown, while ligand blocking antagonist antibodies are included for comparison.

Example 5 - A surrogate non-ligand blocking agonist anti-mouse TNFR2 mAb has antitumor effects in vivo

(See also Figs. 11-17 and the above description of these figures.)

Therapeutic effect on different tumor models

To evaluate the in vivo antitumor effect of non-ligand blocking agonist monoclonal anti-TNFR2 antibodies, a mouse surrogate named 5-A05 was tested in vivo in various tumor models using different isotype formats and alone or in combination with anti-PD-1, as described below.

Mice were bred and maintained at local facilities according to home office recommendations. Six- to eight-week-old female BalbC and C57/BL6 mice were provided by Taconic (Bomholt, Denmark) and maintained at the local animal facilities. CT26, MC38, and B16 cells (ATCC) were grown in glutamax-buffered RPMI medium supplemented with 10% FCS. When the cells were semiconfluent, they were detached with trypsin and resuspended in sterile PBS at 10 × 10 ⁶ cells/mL. Mice were injected s.c. with 100 μL of the cell suspension, corresponding to 1 × 10 ⁶ cells/mouse. Three to eight days after injection, depending on the model, mice were treated twice weekly with 10 mg/kg antibody intraperitoneally. (isotype control, 3-F10 or 5-A05) and as shown in the figures. Tumors were measured twice weekly until they reached 15 mm in diameter, at which time mice were sacrificed.

Non-ligand blocking agonist anti-murine TNFR2 mAb 5-A05 exhibited therapeutic antitumor effects in three different tumor models (Figs. 11-14) with a therapeutic effect in the more treatment-sensitive CT26 (Fig. 11) and a tumor growth inhibitory effect in the more treatment-resistant MC38 and B16 (Figs. 12-14).

The antitumor effect of a non-ligand-blocking agonist anti-mouse TNFR2 mAb does not require FcγR binding but is enhanced by such binding

To assess the importance of the Fc-FcγR interaction on the in vivo antitumor effect of the non-ligand blocking agonist anti-TNFR2 murine surrogate mAb 5A05, different Fc formats of this antibody were studied in vivo in the CT26 tumor model as described below.

Mice were bred and maintained as described above. CT26 cells (ATCC) were grown and injected as described above. When tumors reached 3 x 3 mm, mice were injected intraperitoneally with 10 mg/kg antibody twice weekly (isotype control, 5A05 IgG1, 5A05 IgG2a, or 5-A05-N297A (Fc-deficient). Tumors were measured twice weekly until they reached 15 mm in diameter, at which time mice were sacrificed.

Fc-deficient 5-A05-N297A exhibited clear therapeutic activity compared to the isotype control, indicating that Fc engagement is not entirely required for the therapeutic efficacy of this non-ligand blocking agonist anti-mouse TNFR2 mAb (Fig. 11 A and B). Furthermore, both the IgG1 and IgG2a formats exhibited enhanced therapeutic efficacy. However, the IgG1 format, which preferentially binds to inhibitory Fcγ receptors, exhibited superior therapeutic effect, indicating agonism as one of the important mechanisms of action of this anti-mouse TNFR2 mAb (Fig. 11 A-B). This contrasts with the ligand blocking antagonist surrogate antibody 3-F10, which shows no activity in the defective Fc format and shows better activity in the murine IgG2a format, which is known to preferentially bind the activating FcγR.

A non-ligand blocking agonist antibody (5-A05) according to the present invention and a ligand blocking antagonist antibody (3-F10) are included as a reference.

Combined effect with anti-PD-1 mAb

To evaluate the combined in vivo antitumor effect of non-ligand blocking agonist murine monoclonal anti-TNFR2 surrogate mAb (5-A05) with anti-PD-1, the treatment combination was tested in vivo in the MC38 tumor model as described below.

Mice were bred and maintained as described above. MC38 cells (ATCC) were grown and injected as described above. Eight days after injection, mice were injected intraperitoneally twice weekly with 10 mg/kg antibody (isotype control, anti-mouse PD-1, 5-A05, or a combination of anti-mouse PD-1 and 5-A05) and as shown in Fig. 12A–E. Tumors were measured twice weekly until they reached a diameter of 15 mm, at which time mice were sacrificed.

Both anti-mouse PD-1 and non-ligand blocking agonist anti-mouse TNFR2 mAb 5-A05 exhibited therapeutic tumor growth inhibition effects in the MC38 model (Fig. 12 A-E). When anti-PD1 and 5-A05 were combined, tumors were cured in treatment-resistant MC38 (Fig. 12 D-E).

Combined effect with anti-PD-L1 mAb

To evaluate the combined antitumor effect in vivo of agonist anti-TNFR2 mAbs, we additionally combined a murine surrogate (5A05) with anti-PD-L1 for treatment in the MC38 tumor model as described below.

Mice were bred and maintained as described above. MC38 cells (obtained from Dr M Cragg, University of Southampton) were grown and injected as described above. Six days after injection, mice were treated twice with isotype control antibody or 3F10 (days 1 and 4) or four consecutive days with anti-PD-L1 (clone 10F.9G2, Bioxcell) followed by a fifth injection two days later (for a total of five injections on days 1, 2, 3, 4 and 7) or a combination of both. All antibodies were administered at 10 mg/kg intraperitoneally. Tumours were measured with calipers twice weekly until they reached a volume of 2000 mm ³ , at which time mice were sacrificed.

Both anti-mouse PD-L1 and agonist anti-mouse TNFR2 mAb 5A05 showed therapeutic tumor growth inhibition effect in MC38 model (Fig. 13). When anti-PD-L1 and antagonist anti-mouse TNFR2 mAb 5A05 were combined, the antitumor effect was further enhanced (Fig. 13).

Modulation of immune cells in vivo

To investigate the effect of immune cells on tumor in vivo, BalbC mice were inoculated with CT26 cells as described above. After tumors reached approximately 7 x 7 mm, mice were treated with antibodies at 10 mg/kg intraperitoneally as shown in Fig. 15. Mice were treated on days 1, 4, and 7 and stopped on day 8. Tumors were excised, mechanically divided into small pieces and digested using a mixture of collagenase, 100 μg/ml liberase and 100 μg/ml DNase at 37 °C for 2 x 5 min with vortex in between. After filtration through a 70 μm filter, the cell suspension was washed (400 g for 10 min) with PBS containing 10% FBS. Cells were then resuspended in MACS buffer and stained with a panel of antibodies staining for CD45, CD3, CD8, CD4, and CD25 or a panel of antibodies staining for MHCII, F4/80, Ly6C, CD11b, and Ly6G. Before staining, cells were blocked for non-specific binding with 100 g/ml IVIG. Cells were analyzed on a FACS Verse. Murine Tregs were quantified as CD45 ⁺ CD3 ⁺ CD4 ⁺ CD25 ⁺ and TAMs as CD11b ⁺ Ly6G ^– Ly6C ^– F4/80 ⁺ MHCII ⁺ .

As shown in Fig. 15, treatment with the TNFR2 agonist antibody resulted in an increased influx of CD8 ⁺ T cells into the tumor. A weaker trend towards Treg depletion was also observed. Together, this resulted in a significant improvement in the CD8 ⁺ T cell to Treg ratio (Fig. 15C). In addition, the agonist antibody modulates the myeloid compartment by reducing the number of tumor-associated macrophages.

Model MKPK-NOG/SCID

To confirm the in vitro agonist/T cell proliferative activity of the non-ligand blocking agonist anti-TNFR2 mAb 1-F02, we analyzed the ability of this mAb to induce T cell proliferation in the NOG PBMC model in vivo as described below.

Mice were bred and maintained at local facilities according to home office recommendations. Eight-week-old female NOG mice were supplied by Taconic (Bomholt, Denmark) and maintained at the local animal facilities. For the NOG-PBMC (human primary xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and after washing, the cells were resuspended in sterile PBS at 75 × 10 ⁶ cells/mL. NOG mice were injected i.v. with 200 μL of the cell suspension, corresponding to 15 × 10 ⁶ cells/mouse. Two weeks after injection, mice were treated twice (two days apart) with 10 mg/kg of either isotype control, Yervoy, anti-CD25, Campath, 1-F02, or 1-H10 (a ligand blocking, antagonist anti-TNFR2 mAb). Mouse spleens were harvested 2 days after the last injection. Human T cell subsets were identified and quantified by FACS using the following markers: CD45, CD3, CD4, CD8, CD25, CD127 (all from BD Biosciences). In a separate experiment, spleens from untreated human PBMCs were sacrificed to determine TNFR2 expression on human T cells by FACS (Fig. 16). These FACS data showed that TNFR2 expression on Tregs and CD8+ T cells was highly comparable between human T cells grown and activated in vivo in NOG mice and T cells from human tumors.

1-F02 induced T cell proliferation consistent with that observed in vitro and in vivo with a non-ligand blocking agonist anti-mouse surrogate TNFR2 mAb, whereas the antagonist 1-H10 did not induce proliferation (Fig. 17).

Thus, example 5 shows that:

1. Non-ligand blocking agonist antibodies can exert potent antitumor effects in several tumor models.

2. This effect can be enhanced by combination with anti-PD1 antibodies.

3. Non-blocking ligand agonist antibodies do not exhibit an obligate dependence of FcγR on the antitumor effect, but the effect is enhanced by the involvement of primarily inhibitory FcγR.

4. Non-ligand blocking agonist antibodies increase the influx of CD8-positive T cells and decrease the number of Tregs in tumors. In addition, they decrease the number of TAMs in tumors, thereby altering the composition of both T cells and myeloid cells in the tumor.

5. In human tumors, T cells express TNFR2.

6. In a human xenograft model where tumor TNFR2 expression is mimicked on T cells, the non-ligand blocking agonist antibody 1-F02 increases T cell numbers.

Example 6 - Non-Ligand Blocking Agonist Antibodies Do Not Induce Large Amounts of Proinflammatory Cytokines

(See also Figs. 18-19 and the above description of these figures.)

Release of large amounts of proinflammatory cytokines is one of the possible side effects of immunomodulatory antibodies used to treat patients. Therefore, here we measured cytokine release induced by non-ligand blocking agonist antibodies using two different methods. The first is based on antibody stimulation in in vitro cultures and the second is based on xenotransplantation of human immune cells into immunodeficient mice. For in vitro culture, the culture setup has been shown to significantly influence cytokine release (Vessillier et al., J Immunol Methods. 2015 Sep; 424: 43–52). To account for differences in methodologies, three different in vitro culture setups were used according to recent publications.

For cytokine release assays (CRA) in high density cell culture (HDC), PBMCs were cultured at a concentration of 1 × 10 ⁷ cells/ml in serum-free CTL-Test medium (Cell Technology Limited) supplemented with 2 mM glutamine, 1 mM pyruvate, 100 IU/ml penicillin and streptomycin. 2 ml of cell culture were placed in a 12-well plate. After 48 h, 10 μg/ml antibodies were added to 1 × 10 ⁵ pre-incubated PBMCs in a 96-well flat-bottom plate and incubated for 24 h.

Solid Phase (SP) PBMC CRA was performed by coating the wells of a 96-well plate with 1 μg/ml antibody for 1 h. After washing the plate with PBS, 1 x 10 ⁵ PBMC in 200 μl complete medium per well were added and incubated for 48 h.

Cytokine release was also measured after stimulation with 200 μl whole blood of 5 μg/ml antibody for 48 h.

At the end of the incubation period, the plates were centrifuged, the culture supernatant was collected and stored at -20°C. The concentrations of IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-8 and TNF-α were measured using custom-made MSD plates according to the manufacturer's instructions (Meso Scale Discovery, USA).

Thus, non-ligand blocking agonist antibodies induced any significant release of cytokines other than IFN-γ only under any in vitro conditions (data not shown). The positive control antibodies, alemtuzumab and OKT3, did induce cytokines, the most pronounced being IFN-γ. As seen in Fig. 18, non-ligand blocking agonist antibodies failed to induce IFN-γ under two of the three in vitro conditions; furthermore, the 1-F02 antibody failed to induce IFN-γ under any in vitro conditions.

MKPK-NOG tolerability model

To investigate the tolerability of the non-ligand blocking agonist mAb 1-F02 against human TNFR2, we analyzed cytokine release in vivo in the PBMC-NOG model as described below.

Mice were bred and maintained at local facilities according to home office recommendations. Eight-week-old female NOG mice were supplied by Taconic (Bomholt, Denmark) and maintained at the local animal facilities. For the NOG-PBMC (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and after washing, the cells were resuspended in sterile PBS at 125 × 10 ⁶ cells/mL. NOG mice were injected i.v. with 200 μL of the cell suspension, corresponding to 25 × 10 ⁶ cells/mouse. Two weeks after injection, blood samples were collected to analyze the level of “humanization”, meaning the number of human cells in the blood of NOG mice. When the blood consisted of ∼40% human T cells, the mice were considered humanized. Mice were then treated with 10 μg of either Yervoy, anti-CD3 (OKT-3), 1-F02, or an isotype control mAb. Body temperature was measured before antibody injection and 1 hour after injection (Fig. 19 A). As seen in Fig. 19 A, the positive control antibody OKT3 caused a dramatic decrease in body temperature, as previously published and consistent with the toxicity observed in the clinic with this antibody. In contrast, 1-F02 had no effect on body temperature. Five hours after antibody injection, experiments were stopped and blood was collected for cytokine release assay (MSD). The cytokines measured were human IFN-γ, TNF-α, IL-6, and IL1β. Of these, IFN-γ and TNF-α were quantified at a high enough level to be reliable. As seen in Fig. 19 B and C, the positive control antibody OKT3 induced both significant IFN-γ and TNF-α release (consistent with the toxicity observed in the clinic with this antibody), whereas 1-F02-treated mice did not have significant IFN-γ release. However, there was a trend toward increased TNF-α release, although not significant and not as dramatic as for OKT3.

Thus, Example 6 shows that non-ligand blocking TNFR2 agonist antibodies, in this case illustrated by an antibody called 1-F02, do not induce significant levels of cytokine release as measured by several previously published methods. Since cytokine release is a limiting factor for some immunomodulatory antibodies, this indicates an acceptable safety profile in this regard.

Example 7 - Epitopes of Generated TNFR2-Targeted Antibodies

Knockouts in domain construction

In the first set of experiments, DNA constructs encoding various TNFR2 variants lacking one or more of the 4 extracellular domains described in Table 9 were used. In the second set of experiments, DNA constructs encoding TNFR2 variants in which various parts of domain 3 were replaced by the corresponding murine part as described in Table 10 were used. The latter is possible since none of the antibodies cross-reacts with murine TNFR. In both cases, the constructs were purchased from GeneArt (ThermoFisher). The constructs were cloned into an expression vector containing the CMV promoter and the OriP plasmid origin of replication and transiently expressed in suspension-adapted HEK293-EBNA cells.

Table 9. TNFR2 constructs used for transfection where one or more domains have been deleted

Design Description hTNFR2 wild type, human full length TNFR2 (uniprot#P20333) hTNFR2-∆ 1 hTNFR2 with TNFR-Cys 1 domain deleted (amino acid 39-76) hTNFR2-∆ 2 hTNFR2 with TNFR-Cys 2 domain deleted (amino acid 77-118) hTNFR2-∆ 3 hTNFR2 with TNFR domain deleted-Cys 3 (amino acid 119-162) hTNFR2-∆ 4 hTNFR2 with TNFR domain deleted-Cys 4 (amino acid 163-201) hTNFR2-∆ 1 + 3 hTNFR2 with deleted TNFR-Cys domain 1 and 3 (amino acid 39-76 and 119-162) hTNFR2-∆ 2 + 4 hTNFR2 with deleted TNFR-Cys domain 2 and 4 (amino acid 77-118 and 163-201)

Table 10. TNFR2 constructs used for transfection replaced various parts of domain 3 with the corresponding murine sequences

Design Description hTNFR2 wild type, human full length TNFR2 (uniprot#P20333) mTNFR2 wild type, full length mouse TNFR2 (uniprot #P25119) hTNFR2-m1 hTNFR2 with amino acids 119-132 replaced by amino acids 120-133 from mTNFR2 hTNFR2-m2 hTNFR2 with amino acids 134-144 replaced by amino acids 135-146 from mTNFR2 hTNFR2-m3 hTNFR2 with amino acids 151-160 replaced by amino acids 153-162 from mTNFR2 hTNFR2-m4 hTNFR2 with amino acids 130-144 replaced by amino acids 131-146 from mTNFR2

Flow cytometry based binding assay

HEK-293-E cells were transfected with the corresponding TNFR2 variant cDNA plasmids using Lipofectamine 2000. At 48 h post-transfection, cells were harvested and stained with the indicated antibodies for 30 min. After 2 PBS wash steps, surface-bound antibodies were stained with secondary anti-APC-linked IgG. Cells were washed and stained for live/dead cells prior to flow cytometry analysis on a BD-Verse flow cytometer.

Flow cytometry-based binding experiments on transfected HEK 293 cells clearly showed that domain 1 and domain 2 either do not affect binding (domain 1) or only marginally affect binding (domain 2) of any of the antibodies to these cells. A polyclonal antibody against human TNFR2 was used as a positive control. The positive control antibody showed high binding to all constructs tested, whereas the negative antibody showed no binding (Fig. 20). All antibodies tested showed complete loss of binding to TNFR2 lacking domain 3. Likewise, most antibodies were unable to bind TNFR2 if domain 4 was missing. All antagonist antibodies (1H10, 4H02, and 5B08) showed dramatically reduced binding to TNFR2 Δ4 by more than 50% compared to binding to TNFR2 Δ1 and TNFR2 Δ2. Similarly, deletion of two domains from TNFR2 clearly showed that the absence of domain 3 or 4 significantly abolished binding of all antibodies tested to TNFR2, with the possible exception of the agonist antibody 1F06, while the absence of domain 4 abolished binding of agonist antibodies and reduced binding of antagonist antibodies significantly (Fig. 20 E and F).

Binding to chimeric mouse-human TNFR2

To further narrow the binding site and define epitopes, portions of domain 3 of human TNFR2 were replaced with the corresponding mouse sequence. Since all antibodies exhibit very little cross-reactivity with mouse TNFR2, loss of binding to certain constructs will improve the binding epitope. Figure 21 shows various mouse-human TNFR2 chimeric constructs. Four different substitutions were made, replacing 14 (m1), 12 (m2), 10 (m3), or 16 (m4) amino acids from the human sequence with the corresponding mouse sequence. The remaining three domains (1, 2, 4) contain exclusively human sequences.

These constructs (TNFR2 domains 1-4 with mutations in 3) were then transfected into HEK293 cells and the antibodies were tested for binding using a flow cytometric approach. Polyclonal antibodies against mouse TNFR2 as well as against human TNFR2 were used as positive controls. As expected, due to sequence similarity, both polyclonal control antibodies showed significant cross-reactivity and recognized both human and mouse TNFR2. Clearly, the best signals were obtained when the antibodies were aligned with the intended target.

Our monoclonal antibodies showed strong binding to human TNFR2 but no or very weak binding to murine TNFR2 (Fig. 22, left panel). Similar binding with very little restoration was observed for all clones of the hTNFR2 m1 construct with mutations at amino acids 119-132, indicating that none of the antibodies bind to an epitope in this region. However, mutations at amino acids 134-144 (hTNFR2 m2 construct) completely abolished binding for half of the antibodies tested, corresponding to the antagonist blocking antibodies 1-H10, 4-H02, and 5-B08, indicating that the antibodies bind at least partially within this region. 1-G10 is a partial blocker also strongly affected by this substitution. Notably, agonist antibodies (1-F02, 1-F06, and 4-E08) retained binding using construct 2, strongly suggesting a different epitope compared to the antagonist antibodies. Interestingly, all antibodies lost binding to the hTNFR2 m3 construct with mutations at amino acids 151–160, indicating that all antibodies, both agonists and antagonists, share a partial epitope in this sequence. Testing of the slightly larger hTNFR2 m4 construct with mutations at amino acids 130–144 showed similar binding to the hTNFR2 m2 construct.

Epitope binding conclusions

Grouping the antibodies by their functional role, the agonist antibodies (1-F02, 1-F06, and 4-E08) appear to bind the very distal C-terminal portion of domain 3, spanning amino acids 151-160 of domain 4, whereas the epitope for the antagonists (1-H10, 5-B08, and 4-H02) is shifted more toward the center of domain 3, spanning amino acids 134-160 and likely covering a smaller portion of domain 4. However, despite this, their epitopes appear to overlap to some extent.

None of the antibodies bind to the N-terminal portion of domain 3, amino acids 119-134. Domain 4 binding sites are likely for all antibodies but have not been fully identified.

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SEQUENCE LIST

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<120> NEW MOLECULES OF AGONISTIC ANTI-TNFR2 ANTIBODIES

<130>BI85 PCT

<150> EP 18203996.6

<151> 2018-11-01

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Ser Ala Ile Ser Gly Gly Ala Thr Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Gly Thr Gly Asp Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 16

<211> 112

<212> PRT

<213> Homo sapiens

<400> 16

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Arg Asp Asp Gly

85 90 95

Leu Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 17

<211> 14

<212> PRT

<213> Homo sapiens

<400> 17

Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 18

<211> 19

<212> PRT

<213> Homo sapiens

<400> 18

Ser Ser Ile Ser Ser Ala Ser Gly Tyr Ile Tyr Tyr Gly Asp Ser Val

1 5 10 15

LysGlyArg

<210> 19

<211> 12

<212> PRT

<213> Homo sapiens

<400> 19

Ala Arg Gly Thr Leu Tyr Gly Asp Phe Asp Glu Phe

1 5 10

<210> 20

<211> 14

<212> PRT

<213> Homo sapiens

<400> 20

Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn

1 5 10

<210> 21

<211> 7

<212> PRT

<213> Homo sapiens

<400> 21

Gly Asn Thr Asn Arg Pro Ser

1 5

<210> 22

<211> 13

<212> PRT

<213> Homo sapiens

<400> 22

Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Tyr Val Val

1 5 10

<210> 23

<211> 119

<212> PRT

<213> Homo sapiens

<400> 23

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ala Ser Gly Tyr Ile Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Leu Tyr Gly Asp Phe Asp Glu Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 24

<211> 112

<212> PRT

<213> Homo sapiens

<400> 24

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Gly Tyr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 25

<211> 14

<212> PRT

<213> Homo sapiens

<400> 25

Phe Ser Ser Asn Glu Met Ser Trp Ile Arg Gln Ala Pro Gly

1 5 10

<210> 26

<211> 18

<212> PRT

<213> Homo sapiens

<400> 26

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly Arg

<210> 27

<211> 12

<212> PRT

<213> Homo sapiens

<400> 27

Ala Arg Arg Glu Gly Trp Leu Val Pro Phe Asp Tyr

1 5 10

<210> 28

<211> 14

<212> PRT

<213> Homo sapiens

<400> 28

Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Homo sapiens

<400> 29

Gly Asn Ile Ile Arg Pro Ser

1 5

<210> 30

<211> 13

<212> PRT

<213> Homo sapiens

<400> 30

Cys Gln Ser Phe Asp Thr Thr Leu Ser Gly Ser Ile Val

1 5 10

<210> 31

<211> 118

<212> PRT

<213> Homo sapiens

<400> 31

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn

20 25 30

Glu Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Arg Glu Gly Trp Leu Val Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 32

<211> 112

<212> PRT

<213> Homo sapiens

<400> 32

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Ile Ile Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Thr Thr Leu

85 90 95

Ser Gly Ser Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 33

<211> 14

<212> PRT

<213> Homo sapiens

<400> 33

Phe Ser Arg Tyr Trp Met His Trp Val Arg Gln Val Pro Gly

1 5 10

<210> 34

<211> 19

<212> PRT

<213> Homo sapiens

<400> 34

Ser Gly Ile Ser Asp Ser Gly Val Val Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 35

<211> 10

<212> PRT

<213> Homo sapiens

<400> 35

Ala Arg Ala Gln Ser Val Ala Phe Asp Ile

1 5 10

<210> 36

<211> 15

<212> PRT

<213> Homo sapiens

<400> 36

Cys Ser Gly Ser Ser Ser Asn Ile Gly Ala Gly His Asp Val His

1 5 10 15

<210> 37

<211> 7

<212> PRT

<213> Homo sapiens

<400> 37

Tyr Asp Asp Leu Leu Pro Ser

1 5

<210> 38

<211> 12

<212> PRT

<213> Homo sapiens

<400> 38

Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val

1 5 10

<210> 39

<211> 117

<212> PRT

<213> Homo sapiens

<400> 39

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Asp Ser Gly Val Val Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Gln Ser Val Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 40

<211> 112

<212> PRT

<213> Homo sapiens

<400> 40

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

His Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser

85 90 95

Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 41

<211> 14

<212> PRT

<213> Homo sapiens

<400> 41

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 42

<211> 19

<212> PRT

<213> Homo sapiens

<400> 42

Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ala Val

1 5 10 15

LysGlyArg

<210> 43

<211> 10

<212> PRT

<213> Homo sapiens

<400> 43

Thr Thr Asp Ser Gly Ser Gly Ser Tyr Leu

1 5 10

<210> 44

<211> 15

<212> PRT

<213> Homo sapiens

<400> 44

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10 15

<210> 45

<211> 7

<212> PRT

<213> Homo sapiens

<400> 45

Ser Asn Asn Gln Arg Pro Ser

1 5

<210> 46

<211> 12

<212> PRT

<213> Homo sapiens

<400> 46

Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 47

<211> 117

<212> PRT

<213> Homo sapiens

<400> 47

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Thr Asp Ser Gly Ser Gly Ser Tyr Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 48

<211> 112

<212> PRT

<213> Homo sapiens

<400> 48

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser

85 90 95

Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 49

<211> 14

<212> PRT

<213> Homo sapiens

<400> 49

Phe Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 50

<211> 19

<212> PRT

<213> Homo sapiens

<400> 50

Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 51

<211> 9

<212> PRT

<213> Homo sapiens

<400> 51

Ala Arg Asp Arg Gly Trp Phe Asp Pro

1 5

<210> 52

<211> 14

<212> PRT

<213> Homo sapiens

<400> 52

Cys Ser Gly Ser Arg Ser Asn Ile Asp Asn Ser Tyr Val Ser

1 5 10

<210> 53

<211> 7

<212> PRT

<213> Homo sapiens

<400> 53

Arg Asn Asn Gln Arg Pro Ser

1 5

<210> 54

<211> 12

<212> PRT

<213> Homo sapiens

<400> 54

Cys Ala Thr Trp Asp Asp Ser Leu Ser Gly Pro Val

1 5 10

<210> 55

<211> 116

<212> PRT

<213> Homo sapiens

<400> 55

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 56

<211> 111

<212> PRT

<213> Homo sapiens

<400> 56

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Asp Asn Ser

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu

85 90 95

Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 57

<211> 14

<212> PRT

<213> Homo sapiens

<400> 57

Phe Ser Arg His Ala Met Asn Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 58

<211> 19

<212> PRT

<213> Homo sapiens

<400> 58

Ser Ser Ile Ser Thr Gly Ser Ser Tyr Ile Asp Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 59

<211> 13

<212> PRT

<213> Homo sapiens

<400> 59

Ala Arg Glu Lys Gly His Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 60

<211> 15

<212> PRT

<213> Homo sapiens

<400> 60

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10 15

<210> 61

<211> 7

<212> PRT

<213> Homo sapiens

<400> 61

Gly Asn Ser Tyr Arg Pro Ser

1 5

<210> 62

<211> 13

<212> PRT

<213> Homo sapiens

<400> 62

Cys Gln Ser Tyr Asp Thr Ser Leu Ser Ala Tyr Val Val

1 5 10

<210> 63

<211> 120

<212> PRT

<213> Homo sapiens

<400> 63

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg His

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Thr Gly Ser Ser Tyr Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Lys Gly His Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 64

<211> 113

<212> PRT

<213> Homo sapiens

<400> 64

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Ser Tyr Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Thr Ser

85 90 95

Leu Ser Ala Tyr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

Gly

<210> 65

<211> 14

<212> PRT

<213> Homo sapiens

<400> 65

Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 66

<211> 19

<212> PRT

<213> Homo sapiens

<400> 66

Ser Ala Ile Ser Val Ser Gly Ile Asn Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 67

<211> 11

<212> PRT

<213> Homo sapiens

<400> 67

Ala Arg Asp Thr Gly Ser Leu Gly Val Asp Tyr

1 5 10

<210> 68

<211> 14

<212> PRT

<213> Homo sapiens

<400> 68

Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn

1 5 10

<210> 69

<211> 7

<212> PRT

<213> Homo sapiens

<400> 69

Arg Asn Asn Gln Arg Pro Ser

1 5

<210> 70

<211> 12

<212> PRT

<213> Homo sapiens

<400> 70

Cys Gln Ser Tyr Asp Ser Ser Leu Ser Ile Ser Val

1 5 10

<210> 71

<211> 118

<212> PRT

<213> Homo sapiens

<400> 71

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Val Ser Gly Ile Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Thr Gly Ser Leu Gly Val Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 72

<211> 111

<212> PRT

<213> Homo sapiens

<400> 72

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Ile Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 73

<211> 14

<212> PRT

<213> Homo sapiens

<400> 73

Ser Ser Ser Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 74

<211> 19

<212> PRT

<213> Homo sapiens

<400> 74

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 75

<211> 11

<212> PRT

<213> Homo sapiens

<400> 75

Ala Arg Glu Tyr Ser Gly Tyr Glu Phe Asp Phe

1 5 10

<210> 76

<211> 15

<212> PRT

<213> Homo sapiens

<400> 76

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Arg Ser Asp Val His

1 5 10 15

<210> 77

<211> 7

<212> PRT

<213> Homo sapiens

<400> 77

Gly Asn Arg Asn Arg Pro Ser

1 5

<210> 78

<211> 13

<212> PRT

<213> Homo sapiens

<400> 78

Cys Gln Ser Phe Asp Arg Gly Leu Ser Gly Ser Ile Val

1 5 10

<210> 79

<211> 118

<212> PRT

<213> Homo sapiens

<400> 79

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Tyr Ser Gly Tyr Glu Phe Asp Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 80

<211> 113

<212> PRT

<213> Homo sapiens

<400> 80

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Arg

20 25 30

Ser Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Arg Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Arg Gly

85 90 95

Leu Ser Gly Ser Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

Gly

<210> 81

<211> 14

<212> PRT

<213> Homo sapiens

<400> 81

Ser Ser Ser Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 82

<211> 19

<212> PRT

<213> Homo sapiens

<400> 82

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 83

<211> 11

<212> PRT

<213> Homo sapiens

<400> 83

Ala Arg Glu Tyr Ser Gly Tyr Glu Phe Asp Phe

1 5 10

<210> 84

<211> 15

<212> PRT

<213> Homo sapiens

<400> 84

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Arg Ser Asp Val His

1 5 10 15

<210> 85

<211> 7

<212> PRT

<213> Homo sapiens

<400> 85

Gly Asn Arg Asn Arg Pro Ser

1 5

<210> 86

<211> 13

<212> PRT

<213> Homo sapiens

<400> 86

Cys Gln Ser Phe Asp Arg Gly Leu Ser Gly Ser Ile Val

1 5 10

<210> 87

<211> 118

<212> PRT

<213> Homo sapiens

<400> 87

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Tyr Ser Gly Tyr Glu Phe Asp Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 88

<211> 113

<212> PRT

<213> Homo sapiens

<400> 88

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Arg

20 25 30

Ser Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Arg Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Arg Gly

85 90 95

Leu Ser Gly Ser Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

Gly

<210> 89

<211> 14

<212> PRT

<213> Homo sapiens

<400> 89

Phe Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 90

<211> 19

<212> PRT

<213> Homo sapiens

<400> 90

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 91

<211> 11

<212> PRT

<213> Homo sapiens

<400> 91

Ala Arg Asp Arg Gly Arg Thr Gly Thr Asp Tyr

1 5 10

<210> 92

<211> 14

<212> PRT

<213> Homo sapiens

<400> 92

Cys Ser Gly Thr Thr Ser Asn Ile Gly Ser Tyr Ala Val Asn

1 5 10

<210> 93

<211> 7

<212> PRT

<213> Homo sapiens

<400> 93

Gly Asn Ile Asn Arg Pro Ser

1 5

<210> 94

<211> 12

<212> PRT

<213> Homo sapiens

<400> 94

Cys Gln Ser Tyr Asp Ser Ser Leu Ser Ala Ser Leu

1 5 10

<210> 95

<211> 118

<212> PRT

<213> Homo sapiens

<400> 95

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Arg Thr Gly Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 96

<211> 111

<212> PRT

<213> Homo sapiens

<400> 96

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Thr Thr Ser Asn Ile Gly Ser Tyr

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Ala Ser Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 97

<211> 14

<212> PRT

<213> Homo sapiens

<400> 97

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 98

<211> 19

<212> PRT

<213> Homo sapiens

<400> 98

Ser Thr Ile Ile Gly Ser Gly Ala Asn Thr Trp Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 99

<211> 13

<212> PRT

<213> Homo sapiens

<400> 99

Ala Arg His Glu Gly Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 100

<211> 15

<212> PRT

<213> Homo sapiens

<400> 100

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Val Val His

1 5 10 15

<210> 101

<211> 7

<212> PRT

<213> Homo sapiens

<400> 101

Gly Asn Ser Asn Arg Pro Ser

1 5

<210> 102

<211> 12

<212> PRT

<213> Homo sapiens

<400> 102

Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Arg Val

1 5 10

<210> 103

<211> 120

<212> PRT

<213> Homo sapiens

<400> 103

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ile Gly Ser Gly Ala Asn Thr Trp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Glu Gly Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 104

<211> 112

<212> PRT

<213> Homo sapiens

<400> 104

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser

85 90 95

Leu Asn Gly Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 105

<211> 14

<212> PRT

<213> Homo sapiens

<400> 105

Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 106

<211> 18

<212> PRT

<213> Homo sapiens

<400> 106

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly Arg

<210> 107

<211> 16

<212> PRT

<213> Homo sapiens

<400> 107

Cys Ala Arg Asp Arg Ser Ser Ser Trp Tyr Arg Asp Gly Met Asp Val

1 5 10 15

<210> 108

<211> 15

<212> PRT

<213> Homo sapiens

<400> 108

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10 15

<210> 109

<211> 7

<212> PRT

<213> Homo sapiens

<400> 109

Gly Asn Ser Asn Arg Pro Ser

1 5

<210> 110

<211> 12

<212> PRT

<213> Homo sapiens

<400> 110

Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val

1 5 10

<210> 111

<211> 121

<212> PRT

<213> Homo sapiens

<400> 111

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Arg Ser Ser Ser Trp Tyr Arg Asp Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 112

<211> 112

<212> PRT

<213> Homo sapiens

<400> 112

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser

85 90 95

Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 113

<211> 14

<212> PRT

<213> Homo sapiens

<400> 113

Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 114

<211> 18

<212> PRT

<213> Homo sapiens

<400> 114

Ser Thr Ile Tyr Ser Gly Asp Asn Ala Tyr Tyr Gly Ala Ser Val Arg

1 5 10 15

Gly Arg

<210> 115

<211> 15

<212> PRT

<213> Homo sapiens

<400> 115

Ala Arg Val Tyr Ser Ser Ser Trp Arg Lys Arg Ala Phe Asp Ile

1 5 10 15

<210> 116

<211> 14

<212> PRT

<213> Homo sapiens

<400> 116

Cys Ser Gly Thr Ser Ser Asn Ile Glu Ser Asn Thr Val Asn

1 5 10

<210> 117

<211> 7

<212> PRT

<213> Homo sapiens

<400> 117

Ser Asp Asn Gln Arg Pro Ser

1 5

<210> 118

<211> 12

<212> PRT

<213> Homo sapiens

<400> 118

Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val

1 5 10

<210> 119

<211> 121

<212> PRT

<213> Homo sapiens

<400> 119

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Tyr Ser Gly Asp Asn Ala Tyr Tyr Gly Ala Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Tyr Ser Ser Ser Trp Arg Lys Arg Ala Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 120

<211> 111

<212> PRT

<213> Homo sapiens

<400> 120

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn Ile Glu Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 121

<211> 14

<212> PRT

<213> Homo sapiens

<400> 121

Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly

1 5 10

<210> 122

<211> 19

<212> PRT

<213> Homo sapiens

<400> 122

Ala Leu Ile Trp Tyr Asp Gly Gly Asn Glu Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 123

<211> 11

<212> PRT

<213> Homo sapiens

<400> 123

Val Arg Glu Thr Gly Asn Tyr Gly Met Asp Val

1 5 10

<210> 124

<211> 15

<212> PRT

<213> Homo sapiens

<400> 124

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10 15

<210> 125

<211> 7

<212> PRT

<213> Homo sapiens

<400> 125

Arg Asn Asn Gln Arg Pro Ser

1 5

<210> 126

<211> 12

<212> PRT

<213> Homo sapiens

<400> 126

Cys Ala Thr Trp Asp Asp Arg Val Asn Gly Pro Val

1 5 10

<210> 127

<211> 118

<212> PRT

<213> Homo sapiens

<400> 127

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Trp Tyr Asp Gly Gly Asn Glu Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Glu Thr Gly Asn Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 128

<211> 112

<212> PRT

<213> Homo sapiens

<400> 128

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Arg

85 90 95

Val Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 129

<211> 14

<212> PRT

<213> Homo sapiens

<400> 129

Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly

1 5 10

<210> 130

<211> 19

<212> PRT

<213> Homo sapiens

<400> 130

Ala Leu Ile Trp Tyr Asp Gly Gly Asn Glu Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 131

<211> 11

<212> PRT

<213> Homo sapiens

<400> 131

Ala Arg Tyr Tyr Gly Asp Gly Gly Phe Asp Pro

1 5 10

<210> 132

<211> 15

<212> PRT

<213> Homo sapiens

<400> 132

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Val Val His

1 5 10 15

<210> 133

<211> 7

<212> PRT

<213> Homo sapiens

<400> 133

Ser Asn Asn Gln Arg Pro Ser

1 5

<210> 134

<211> 12

<212> PRT

<213> Homo sapiens

<400> 134

Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 135

<211> 118

<212> PRT

<213> Homo sapiens

<400> 135

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Ser Tyr Asp Gly Gly Gly Lys Tyr Phe Ala Asp Pro Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Gly Asp Gly Gly Phe Asp Pro Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 136

<211> 112

<212> PRT

<213> Homo sapiens

<400> 136

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser

85 90 95

Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 137

<211> 14

<212> PRT

<213> Homo sapiens

<400> 137

Phe Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 138

<211> 19

<212> PRT

<213> Homo sapiens

<400> 138

Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 139

<211> 8

<212> PRT

<213> Homo sapiens

<400> 139

Ala Lys Asp Pro Leu Phe Asp Ser

1 5

<210> 140

<211> 15

<212> PRT

<213> Homo sapiens

<400> 140

Cys Thr Gly Arg Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10 15

<210> 141

<211> 7

<212> PRT

<213> Homo sapiens

<400> 141

Asp Asn Asn Lys Arg Pro Ser

1 5

<210> 142

<211> 12

<212> PRT

<213> Homo sapiens

<400> 142

Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 143

<211> 115

<212> PRT

<213> Homo sapiens

<400> 143

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Pro Leu Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 144

<211> 112

<212> PRT

<213> Homo sapiens

<400> 144

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Arg Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser

85 90 95

Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 145

<211> 14

<212> PRT

<213> Homo sapiens

<400> 145

Phe Asn Thr Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 146

<211> 18

<212> PRT

<213> Homo sapiens

<400> 146

Ser Val Leu Tyr Ser Asp Asp Asp Thr His Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly Arg

<210> 147

<211> 17

<212> PRT

<213> Homo sapiens

<400> 147

Ala Arg Asp Cys Gly Gly Asp Cys His Ser Gly Asp Asp Ala Phe Asp

1 5 10 15

Ile

<210> 148

<211> 14

<212> PRT

<213> Homo sapiens

<400> 148

Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn

1 5 10

<210> 149

<211> 7

<212> PRT

<213> Homo sapiens

<400> 149

Asp Asn Asp Lys Arg Pro Ser

1 5

<210> 150

<211> 12

<212> PRT

<213> Homo sapiens

<400> 150

Cys Ala Ala Trp His Asp Ser Leu Asn Gly Trp Val

1 5 10

<210> 151

<211> 123

<212> PRT

<213> Homo sapiens

<400> 151

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Leu Tyr Ser Asp Asp Asp Thr His Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Cys Gly Gly Asp Cys His Ser Gly Asp Asp Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 152

<211> 111

<212> PRT

<213> Homo sapiens

<400> 152

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asp Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp His Asp Ser Leu

85 90 95

Asn Gly Trp Val Leu Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 153

<211> 14

<212> PRT

<213> Homo sapiens

<400> 153

Phe Ser Ala Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 154

<211> 19

<212> PRT

<213> Homo sapiens

<400> 154

Ala Val Val Ser Tyr Asp Gly Arg Glu Lys His Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 155

<211> 14

<212> PRT

<213> Homo sapiens

<400> 155

Ala Arg Ser Asp Gly Gly Tyr Asp Ser Asp Ser Gly Tyr Tyr

1 5 10

<210> 156

<211> 14

<212> PRT

<213> Homo sapiens

<400> 156

Cys Ser Gly Ser Thr Ser Asn Ile Gly Ser Asn Phe Val Tyr

1 5 10

<210> 157

<211> 7

<212> PRT

<213> Homo sapiens

<400> 157

Asp Asn Asn Lys Arg Pro Ser

1 5

<210> 158

<211> 11

<212> PRT

<213> Homo sapiens

<400> 158

Cys Ser Ser Tyr Ala Tyr Ser Asp Asn Ile Leu

1 5 10

<210> 159

<211> 121

<212> PRT

<213> Homo sapiens

<400> 159

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Val Ser Tyr Asp Gly Arg Glu Lys His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Gly Gly Tyr Asp Ser Asp Ser Gly Tyr Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 160

<211> 110

<212> PRT

<213> Homo sapiens

<400> 160

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Ser Asn

20 25 30

Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Tyr Ser Asp

85 90 95

Asn Ile Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 161

<211> 14

<212> PRT

<213> Homo sapiens

<400> 161

Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 162

<211> 19

<212> PRT

<213> Homo sapiens

<400> 162

Ser Gly Ile Ser Ser Ser Gly Ser Ser Ala Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 163

<211> 18

<212> PRT

<213> Homo sapiens

<400> 163

Ala Arg His Tyr Tyr Tyr His Ile Ala Gly Tyr Tyr Tyr Asp Thr Phe

1 5 10 15

Asp Ile

<210> 164

<211> 14

<212> PRT

<213> Homo sapiens

<400> 164

Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn Thr Val Asn

1 5 10

<210> 165

<211> 7

<212> PRT

<213> Homo sapiens

<400> 165

Gly Asn Thr Asn Arg Pro Ser

1 5

<210> 166

<211> 12

<212> PRT

<213> Homo sapiens

<400> 166

Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Val Val

1 5 10

<210> 167

<211> 125

<212> PRT

<213> Homo sapiens

<400> 167

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Ser Ser Gly Ser Ser Ala Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Tyr Tyr Tyr His Ile Ala Gly Tyr Tyr Tyr Asp Thr Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 168

<211> 111

<212> PRT

<213> Homo sapiens

<400> 168

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 169

<211> 14

<212> PRT

<213> Homo sapiens

<400> 169

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 170

<211> 19

<212> PRT

<213> Homo sapiens

<400> 170

Ala Thr Ile Ser Tyr His Gly Ser Asp Lys Asp Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 171

<211> 18

<212> PRT

<213> Homo sapiens

<400> 171

Ala Arg Asp Ala Asn Tyr His Ser Ser Gly Tyr Tyr Tyr Asp Val Phe

1 5 10 15

Asp Ile

<210> 172

<211> 14

<212> PRT

<213> Homo sapiens

<400> 172

Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn

1 5 10

<210> 173

<211> 7

<212> PRT

<213> Homo sapiens

<400> 173

Gly Asn Ser Asn Arg Pro Ser

1 5

<210> 174

<211> 12

<212> PRT

<213> Homo sapiens

<400> 174

Cys Ala Ala Trp Asp Asp Ser Leu Ser Thr Trp Val

1 5 10

<210> 175

<211> 125

<212> PRT

<213> Homo sapiens

<400> 175

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Tyr His Gly Ser Asp Lys Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ala Asn Tyr His Ser Ser Gly Tyr Tyr Tyr Asp Val Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 176

<211> 111

<212> PRT

<213> Homo sapiens

<400> 176

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 177

<211> 14

<212> PRT

<213> Homo sapiens

<400> 177

Phe Ser Asp Tyr Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly

1 5 10

<210> 178

<211> 19

<212> PRT

<213> Homo sapiens

<400> 178

Ser Gly Ile Ser Gly Ser Gly Gly Tyr Ile His Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 179

<211> 11

<212> PRT

<213> Homo sapiens

<400> 179

Ala Arg Glu Gly Leu Leu Pro Asp Ala Phe Asp

1 5 10

<210> 180

<211> 14

<212> PRT

<213> Homo sapiens

<400> 180

Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser

1 5 10

<210> 181

<211> 7

<212> PRT

<213> Homo sapiens

<400> 181

Arg Asn Asn Gln Arg Pro Ser

1 5

<210> 182

<211> 12

<212> PRT

<213> Homo sapiens

<400> 182

Cys Ala Ala Trp Asp Asp Ser Val Ser Gly Trp Val

1 5 10

<210> 183

<211> 119

<212> PRT

<213> Homo sapiens

<400> 183

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Gly Ser Gly Gly Tyr Ile His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Leu Leu Pro Asp Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 184

<211> 111

<212> PRT

<213> Homo sapiens

<400> 184

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Val

85 90 95

Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 185

<211> 14

<212> PRT

<213> Homo sapiens

<400> 185

Phe Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 186

<211> 19

<212> PRT

<213> Homo sapiens

<400> 186

Ala Val Met Ser Tyr Asp Glu Tyr Asn Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 187

<211> 13

<212> PRT

<213> Homo sapiens

<400> 187

Ala Lys Gly Phe Tyr Gly Asp Tyr Pro Leu Trp Asp Tyr

1 5 10

<210> 188

<211> 14

<212> PRT

<213> Homo sapiens

<400> 188

Cys Ser Gly Gly Asn Ser Asn Ile Gly Thr Asn Thr Val Asp

1 5 10

<210> 189

<211> 7

<212> PRT

<213> Homo sapiens

<400> 189

Ser Asn Asn Gln Arg Pro Ser

1 5

<210> 190

<211> 12

<212> PRT

<213> Homo sapiens

<400> 190

Cys Ala Ala Trp Asp Asp Ser Val Asn Gly Pro Val

1 5 10

<210> 191

<211> 120

<212> PRT

<213> Homo sapiens

<400> 191

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Met Ser Tyr Asp Glu Tyr Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Phe Tyr Gly Asp Tyr Pro Leu Trp Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 192

<211> 111

<212> PRT

<213> Homo sapiens

<400> 192

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Gly Asn Ser Asn Ile Gly Thr Asn

20 25 30

Thr Val Asp Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Val

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 193

<211> 14

<212> PRT

<213> Homo sapiens

<400> 193

Phe Ser Ser Tyr Glu Met Asn Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 194

<211> 18

<212> PRT

<213> Homo sapiens

<400> 194

Ser Thr Ile Thr Gly Gly Gly Ser Ile Tyr Asp Ala Asn Ser Val Gln

1 5 10 15

Gly Arg

<210> 195

<211> 18

<212> PRT

<213> Homo sapiens

<400> 195

Ala Arg Asp Ser Thr Tyr His Ser Ser Gly Tyr Tyr Tyr Asp Val Phe

1 5 10 15

Asp Ile

<210> 196

<211> 14

<212> PRT

<213> Homo sapiens

<400> 196

Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn

1 5 10

<210> 197

<211> 7

<212> PRT

<213> Homo sapiens

<400> 197

Gly Asn Ser Asn Arg Pro Ser

1 5

<210> 198

<211> 13

<212> PRT

<213> Homo sapiens

<400> 198

Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly His Trp Val

1 5 10

<210> 199

<211> 124

<212> PRT

<213> Homo sapiens

<400> 199

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Thr Gly Gly Gly Ser Ile Tyr Asp Ala Asn Ser Val Gln

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Ser Thr Tyr His Ser Ser Gly Tyr Tyr Tyr Asp Val Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 200

<211> 112

<212> PRT

<213> Homo sapiens

<400> 200

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 201

<211> 14

<212> PRT

<213> Homo sapiens

<400> 201

Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 202

<211> 20

<212> PRT

<213> Homo sapiens

<400> 202

Ser Ala Val Phe Gly Ser Gly His Gly Asn Thr Phe Tyr Ala Asp Ala

1 5 10 15

Val Lys Gly Arg

20

<210> 203

<211> 14

<212> PRT

<213> Homo sapiens

<400> 203

Ala Arg Glu Gln Leu Trp Phe Gly Gln Asp Ala Phe Asp Ile

1 5 10

<210> 204

<211> 14

<212> PRT

<213> Homo sapiens

<400> 204

Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn

1 5 10

<210> 205

<211> 7

<212> PRT

<213> Homo sapiens

<400> 205

Gly Asn Ser Asn Arg Pro Ser

1 5

<210> 206

<211> 12

<212> PRT

<213> Homo sapiens

<400> 206

Cys Gln Ser Tyr Asp Ser Ser Leu Ser Ala Ser Val

1 5 10

<210> 207

<211> 122

<212> PRT

<213> Homo sapiens

<400> 207

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Val Phe Gly Ser Gly His Gly Asn Thr Phe Tyr Ala Asp Ala

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gln Leu Trp Phe Gly Gln Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 208

<211> 111

<212> PRT

<213> Homo sapiens

<400> 208

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Ala Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 209

<211> 14

<212> PRT

<213> Homo sapiens

<400> 209

Phe Ser Asp Ala Trp Met Thr Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 210

<211> 19

<212> PRT

<213> Homo sapiens

<400> 210

Ser Asp Leu Ser Asp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

LysGlyArg

<210> 211

<211> 11

<212> PRT

<213> Homo sapiens

<400> 211

Gly Arg Leu Ala Ala Gly Gly Pro Val Asp Tyr

1 5 10

<210> 212

<211> 15

<212> PRT

<213> Homo sapiens

<400> 212

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10 15

<210> 213

<211> 7

<212> PRT

<213> Homo sapiens

<400> 213

Ser Asn Asn Gln Arg Pro Ser

1 5

<210> 214

<211> 12

<212> PRT

<213> Homo sapiens

<400> 214

Cys Ser Val Trp Asp Asp Ser Leu Asn Ser Trp Val

1 5 10

<210> 215

<211> 118

<212> PRT

<213> Homo sapiens

<400> 215

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Leu Ser Asp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Leu Ala Ala Gly Gly Pro Val Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 216

<211> 112

<212> PRT

<213> Homo sapiens

<400> 216

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Trp Asp Asp Ser

85 90 95

Leu Asn Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 217

<211> 330

<212> PRT

<213> Homo sapiens

<400> 217

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 218

<211> 105

<212> PRT

<213> Homo sapiens

<400> 218

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 219

<211> 330

<212> PRT

<213> Mus musculus

<400> 219

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

275 280 285

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 220

<211> 330

<212> PRT

<213> Mus musculus

<400> 220

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Ala Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

275 280 285

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 221

<211> 105

<212> PRT

<213> Mus musculus

<400> 221

Gln Pro Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp Phe

20 25 30

Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro Val

35 40 45

Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu Arg

65 70 75 80

His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val Glu

85 90 95

Lys Ser Leu Ser Arg Ala Asp Cys Ser

100 105

<210> 222

<211> 1356

<212> DNA

<213> Homo sapiens

<400> 222

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120

ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagtac atattacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 240

caaatgaaca gcctgagagc cgaggacact gccgtgtatt actgtgcgag agatcgaagc 300

agcagctggt accgcgatgg tatggacgtc tggggccaag gtacactggt caccgtgagc 360

tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420

gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480

tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540

tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 600

acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 660

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag ccccatcga gaaaaccatc 1020

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1080

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acgcagaaga gcctctccct gtctccgggt aaatga 1356

<210> 223

<211> 654

<212> DNA

<213> Homo sapiens

<400> 223

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60

tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtatcagcag 120

ctcccaggaa cggcccccaa actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180

cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 240

cggtccgagg atgaggctga ttattactgt gcagcatggg atgacagcct gagtggttgg 300

gtgttcggcg gaggaaccaa gctgacggtc ctaggtcagc ccaaggctgc cccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc atga 654

<---

Claims (89)

1. An agonist antibody molecule or antigen-binding fragment thereof that specifically binds to TNFR2 on a target cell, wherein said antibody molecule or antigen-binding fragment thereof does not block the binding of a TNF-α ligand to TNFR2, wherein the antibody or antigen-binding fragment thereof is characterized by intrinsic agonist activity, wherein the antibody molecule or antigen-binding fragment thereof comprises: (a) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; or (b) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14; or (c) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22; or (d) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30; or (e) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38; or (f) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46; or (g) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54; or (h) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62; or (i) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70; or (j) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:76, SEQ ID NO:77 and SEQ ID NO:78; or (k) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86; or (l) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94; or (m) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, and a variable light chain (VL) comprising the following CDRs: SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102. 2. An antibody molecule or its antigen-binding fragment according to claim 1, characterized in that it also binds to the Fcγ receptor. 3. An antibody molecule or its antigen-binding fragment according to any one of claims 1, 2, characterized in that it binds with higher affinity to inhibitory Fcγ receptors than to activating Fcγ receptors. 4. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that their binding to TNFR2 leads to a change in the number and/or frequency of cells expressing TNFR2 in the affected tissue. 5. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that their binding to TNFR2 leads to infiltration of T cells and/or myeloid cells into the affected tissue, and/or a change in the composition of T cells and/or myeloid cells in the affected tissue. 6. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-5, characterized in that it is selected from the group consisting of a full-length antibody, a chimeric antibody, a bivalent or polyvalent antibody molecule containing single-chain antibodies, Fab, Fv, scFv, Fab' and/or (Fab') ₂ . 7. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-6, binding to human TNFR2 (hTNFR2) and/or cynomolgus macaque TNFR2 (cmTNFR2). 8. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody molecule is selected from the group consisting of a human IgG antibody molecule, a humanized IgG antibody molecule, and an IgG antibody molecule of human origin. 9. An antibody molecule or antigen-binding fragment thereof according to claim 8, wherein the antibody molecule is a human IgG2 antibody. 10. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody molecule is a monoclonal antibody. 11. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-10, characterized in that it does not bind specifically to an epitope containing or consisting of the KCSPG sequence. 12. An antibody molecule or its antigen-binding fragment according to any one of paragraphs 1-11, characterized in that the antibody molecule or its antigen-binding fragment contains: (a) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:7 and the variable light chain (VL) amino acid sequence of SEQ ID NO:8; or (b) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:15 and the variable light chain (VL) amino acid sequence of SEQ ID NO:16; or (c) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:23 and the variable light chain (VL) amino acid sequence of SEQ ID NO:24; or (d) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:31 and the variable light chain (VL) amino acid sequence of SEQ ID NO:32; or (e) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:39 and the variable light chain (VL) amino acid sequence of SEQ ID NO:40; or (f) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:47 and the variable light chain (VL) amino acid sequence of SEQ ID NO:48; or (g) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:55 and the variable light chain (VL) amino acid sequence of SEQ ID NO:56; or (h) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:63 and the variable light chain (VL) amino acid sequence of SEQ ID NO:64; or (i) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:71 and the variable light chain (VL) amino acid sequence of SEQ ID NO:72; or (j) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:79 and the variable light chain (VL) amino acid sequence of SEQ ID NO:80; or (k) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:87 and the variable light chain (VL) amino acid sequence of SEQ ID NO:88; or (l) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:95 and the variable light chain (VL) amino acid sequence of SEQ ID NO:96; or (m) the amino acid sequence of the variable heavy chain (VH) of SEQ ID NO:103 and the amino acid sequence of the variable light chain (VL) of SEQ ID NO:104. 13. An antibody molecule or its antigen-binding fragment according to any one of paragraphs 1-12, characterized in that the antibody molecule or its antigen-binding fragment contains: (i) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:7, and the variable light chain (VL) amino acid sequence of SEQ ID NO:8; or (ii) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:15, and the variable light chain (VL) amino acid sequence of SEQ ID NO:16; or (iii) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:23, and the variable light chain (VL) amino acid sequence of SEQ ID NO:24; or (iv) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:31, and the variable light chain (VL) amino acid sequence of SEQ ID NO:32; or (v) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:39, and the variable light chain (VL) amino acid sequence of SEQ ID NO:40; or (vi) the variable heavy chain (VH) amino acid sequence of SEQ ID NO:47, and the variable light chain (VL) amino acid sequence of SEQ ID NO:48. 14. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-11, wherein the antibody molecule is an antibody molecule that is capable of competing in binding to TNFR2 with the antibody molecule claimed in any one of claims 12, 13. 15. An isolated nucleic acid encoding an antibody molecule or an antigen-binding fragment thereof as claimed in any one of claims 1 to 14, wherein said nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of the antibody or antigen-binding fragment thereof. 16. An expression plasmid containing the nucleotide sequence of claim 15. 17. A virus for delivering an antibody molecule or an antigen-binding fragment thereof, comprising a nucleic acid encoding said antibody or an antigen-binding fragment thereof, wherein the virus is characterized in that said nucleic acid is a nucleic acid according to claim 15, and said antibody is an antibody or an antigen-binding fragment thereof as defined in any of claims 1-14. 18. The virus according to claim 17, further comprising a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 19. A cell for delivering an antibody molecule or its antigen-binding fragment, as claimed in any one of paragraphs 1-14, containing a nucleic acid according to paragraph 15. 20. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-14, a nucleic acid according to claim 15, a plasmid according to claim 16, a virus according to any one of claims 17, 18 and/or a cell according to claim 19 for use in the treatment of an autoimmune disease characterized by the expression of TNF2 in immune cells. 21. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-14, a nucleic acid according to claim 15, a plasmid according to claim 16, a virus according to any one of claims 17, 18 and/or a cell according to claim 19 for use in the treatment of cancer in a patient having high expression of TNF2 in diseased tissue compared to corresponding healthy tissue. 22. An antibody molecule or antigen-binding fragment thereof according to any one of claims 1-14, a nucleic acid according to claim 15, a plasmid according to claim 16, a virus according to claim 17 and/or a cell according to claim 19 for use in the treatment of cancer in a patient having high expression of TNF2 in diseased tissue compared to corresponding healthy tissue, in combination with: an antibody molecule that specifically binds to a checkpoint inhibitor; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 23. Use of an antibody molecule or antigen-binding fragment thereof according to any one of claims 1-14, a nucleic acid according to claim 15, a plasmid according to claim 16, a virus according to claim 17 and/or a cell according to claim 19 for the production of a pharmaceutical composition used in the treatment of cancer in a patient having high expression of TNF2 in affected tissue compared to the corresponding healthy tissue. 24. Use according to any of paragraphs 22, 23, characterized in that the said pharmaceutical composition is administered in combination with: an antibody molecule that specifically binds to a checkpoint inhibitor; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 25. Use of an antibody molecule or an antigen-binding fragment thereof according to any one of claims 1-14, a nucleic acid according to claim 15, a plasmid according to claim 16, a virus according to claim 17 and/or a cell according to claim 19 for the production of a pharmaceutical composition used in the treatment of an autoimmune disease characterized by the expression of TNF2 in immune cells. 26. Use according to claim 25, characterized in that the pharmaceutical composition is intended for use in the treatment of an autoimmune disease in a patient having a high expression of TNFR2 in the affected tissue compared to the corresponding healthy tissue. 27. Use according to paragraph 25 or 26, characterized in that the said pharmaceutical composition is administered in combination with: an antibody molecule that specifically binds to a checkpoint inhibitor; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 28. A pharmaceutical composition for inducing TNF2 signaling, comprising an antibody molecule or an antigen-binding fragment thereof according to any one of claims 1-14, a nucleic acid according to claim 15, a plasmid according to claim 16, a virus according to claim 17 and/or a cell according to claim 19. 29. The pharmaceutical composition according to claim 28, wherein the composition comprises a pharmaceutically acceptable diluent, carrier, filler and/or excipient. 30. The pharmaceutical composition of claim 28 for use in the treatment of cancer in a patient having high expression of TNF2 in the affected tissue compared to the corresponding healthy tissue, or an autoimmune disease characterized by expression of TNF2 in immune cells. 31. A pharmaceutical composition according to claim 30 for use in the treatment of cancer in a patient having high expression of TNF2 in the affected tissue compared to the corresponding healthy tissue, in combination with a pharmaceutical composition comprising: an antibody molecule that specifically binds to a checkpoint inhibitor; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 32. A method for treating cancer in a subject having high expression of TNF2 in diseased tissue compared to corresponding healthy tissue, comprising administering to the subject a therapeutically effective amount of an antibody molecule or antigen-binding fragment thereof according to any one of claims 1-14, a nucleic acid according to claim 15, a plasmid according to claim 16, a virus according to claim 17, a cell according to claim 19, or a pharmaceutical composition according to claim 28 or 29. 33. A method for treating an autoimmune disease characterized by the expression of TNF2 in immune cells in a subject, comprising administering to the subject a therapeutically effective amount of an antibody molecule or an antigen-binding fragment thereof according to any one of claims 1-14, a nucleic acid according to claim 15, a plasmid according to claim 16, a virus according to claim 17, a cell according to claim 19, or a pharmaceutical composition according to claim 28 or 29. 34. The method of treating cancer according to claim 32, characterized in that a subject having a high expression of TNF2 in the affected tissue compared to the corresponding healthy tissue is also administered a therapeutically effective amount of: an antibody molecule that specifically binds to a checkpoint inhibitor; a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor; and/or a cell containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, a plasmid containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor, or a virus containing a nucleotide sequence encoding an antibody molecule that specifically binds to a checkpoint inhibitor. 35. An antibody molecule or antigen-binding fragment thereof for use according to claim 22, a nucleic acid for use according to claim 22, a plasmid for use according to claim 22, a virus for use according to claim 22, a cell for use according to claim 22, the use according to claim 24 or 27, a pharmaceutical composition according to claim 31 or a method according to claim 34, characterized in that the checkpoint inhibitor is PD-1. 36. An antibody molecule or antigen-binding fragment thereof for use according to claim 22, a nucleic acid for use according to claim 22, a plasmid for use according to claim 22, a virus for use according to claim 22, a cell for use according to claim 22, the use according to claim 24 or 27, a pharmaceutical composition according to claim 31 or a method according to claim 34, characterized in that the checkpoint inhibitor is PD-L1. 37. An antibody molecule or antigen-binding fragment thereof for use according to claims 21, 22 or 35, 36, a nucleic acid for use according to claims 21, 22 or 38, 39, a plasmid for use according to claims 21, 22 or 35, 36, a virus for use according to claims 21, 22 or 35, 36, a cell for use according to claims 21, 22 or claims 35, 36, the use according to claims 23-27 or 35, 36, a pharmaceutical composition according to claims 28, 29, 31 or 35, 36 or the method according to claims 32, 33-36, characterized in that the cancer is a solid cancer.